Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose

ABSTRACT

The present invention is directed to a yeast strain, or strains, secreting a full suite, or any subset of that full suite, of enzymes to hydrolyze corn starch, corn fiber, lignocellulose, (including enzymes that hydrolyze linkages in cellulose, hemicellulose, and between lignin and carbohydrates) and to utilize pentose sugars (xylose and arabinose). The invention is also directed to the set of proteins that are well expressed in yeast for each category of enzymatic activity. The resulting strain, or strains can be used to hydrolyze starch and cellulose simultaneously. The resulting strain, or strains can be also metabolically engineered to produce less glycerol and uptake acetate. The resulting strain, or strains can also be used to produce ethanol from granular starch without liquefaction. The resulting strain, or strains, can be further used to reduce the amount of external enzyme needed to hydrolyze a biomass feedstock during an Simultaneous Saccharification and Fermentation (SSF) process, or to increase the yield of ethanol during SSF at current saccharolytic enzyme loadings. In addition, multiple enzymes of the present invention can be co-expressed in cells of the invention to provide synergistic digestive action on biomass feedstock. In some aspects, host cells expressing different heterologous saccharolytic enzymes can also be co-cultured together and used to produce ethanol from biomass feedstock.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/178,653, filed Feb. 12, 2014, which is a continuation of U.S.application Ser. No. 13/701,652, filed Aug. 7, 2013, which is theNational Stage of International Application Number PCT/US2011/039192,filed Jun. 3, 2011, which claims the benefit of U.S. ProvisionalApplication No. 61/351,165, filed Jun. 3, 2010, and U.S. ProvisionalApplication No. 61/420,142, filed Dec. 6, 2010, each of which areincorporated by reference herein.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:115235-212SeqList.txt; Size: 1,995,410 bytes; and Date of Creation: Nov.6, 2015) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Biomass is biological material from living, or recently livingorganisms, such as wood, waste, (hydrogen) gas, and alcohol fuels.Biomass is carbon, hydrogen and oxygen based. Nitrogen and smallquantities of other atoms, including alkali, alkaline earth and heavymetals can be found as well. Metals are often found in functionalmolecules such as the porphyrins which include chlorophyll whichcontains magnesium. Plants in particular combine water and carbondioxide to sugar building blocks. The required energy is produced fromlight via photosynthesis based on chlorophyll. On average, between 0.1and 1% of the available light is stored as chemical energy in plants.The sugar building blocks are the starting point for all of the majorfractions found in terrestrial plants, lignin, hemicellulose andcellulose. Biomass is widely recognized as a promising source of rawmaterial for production of renewable fuels and chemicals. The primaryobstacle impeding the more widespread production of energy from biomassfeedstocks is the general absence of low-cost technology for overcomingthe recalcitrance of these materials to conversion into useful fuels.Biomass contains carbohydrate fractions (e.g., starch, cellulose, andhemicellulose) that can be converted into ethanol. In order to convertthese fractions, the starch, cellulose, and, hemicellulose mustultimately be converted or hydrolyzed into monosaccharides; it is thehydrolysis that has historically proven to be problematic.

Biologically mediated processes are promising for energy conversion, inparticular for the conversion of biomass into fuels. Biomass processingschemes involving enzymatic or microbial hydrolysis commonly involvefour biologically mediated transformations: (1) the production ofsaccharolytic enzymes (amylases, cellulases and hemicellulases); (2) thehydrolysis of carbohydrate components present in pretreated biomass tosugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose,and galactose); and (4) the fermentation of pentose sugars (e.g., xyloseand arabinose). These four transformations occur in a single step in aprocess configuration called consolidated bioprocessing (CBP), which isdistinguished from other less highly integrated configurations in thatit does not involve a dedicated process step for cellulase and/orhemicellulase production.

CBP offers the potential for lower cost and higher efficiency thanprocesses featuring dedicated saccharolytic enzyme production. Thebenefits result in part from avoided capital costs, substrate and otherraw materials, and utilities associated with saccharolytic enzymeproduction. In addition, several factors support the realization ofhigher rates of hydrolysis, and hence reduced reactor volume and capitalinvestment using CBP, including enzyme-microbe synergy and the use ofthermophilic organisms and/or complexed saccharolytic systems. Moreover,cellulose-adherent cellulolytic microorganisms are likely to competesuccessfully for products of cellulose hydrolysis with non-adheredmicrobes, e.g., contaminants, which could increase the stability ofindustrial processes based on microbial cellulose utilization. Progressin developing CBP-enabling microorganisms is being made through twostrategies: engineering naturally occurring saccharolytic microorganismsto improve product-related properties, such as yield and titer; andengineering non-saccharolytic organisms that exhibit high product yieldsand titers to express a heterologous saccharolytic enzyme systemenabling starch, cellulose, and, hemicellulose utilization.

The breakdown of starch down into sugar requires amylolytic enzymes.Amylase is an example of an amylolytic enzyme that is present in humansaliva, where it begins the chemical process of digestion. The pancreasalso makes amylase (alpha amylase) to hydrolyze dietary starch intodisaccharides and trisaccharides which are converted by other enzymes toglucose to supply the body with energy. Plants and some bacteria alsoproduce amylases. Amylases are glycoside hydrolases and act onα-1,4-glycosidic bonds.

Several amylolytic enzymes are implicated in starch hydrolysis.Alpha-amylases (EC 3.2.1.1) (alternate names: 1,4-α-D-glucanglucanohydrolase; glycogenase) are calcium metalloenzymes, i.e.,completely unable to function in the absence of calcium. By acting atrandom locations along the starch chain, alpha-amylase breaks downlong-chain carbohydrates, ultimately yielding maltotriose and maltosefrom amylose, or maltose, glucose and “limit dextrin” from amylopectin.Because it can act anywhere on the substrate, alpha-amylase tends to befaster-acting than beta-amylase. Another form of amylase, beta-amylase(EC 3.2.1.2) (alternate names: 1,4-α-D-glucan maltohydrolase;glycogenase; saccharogen amylase) catalyzes the hydrolysis of the secondα-1,4 glycosidic bond, cleaving off two glucose units (maltose) at atime. The third amylase is gamma-amylase (EC 3.2.1.3) (alternate names:Glucan 1,4-α-glucosidase; amyloglucosidase; Exo-1,4-α-glucosidase;glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase).In addition to cleaving the last α(1-4)glycosidic linkages at thenonreducing end of amylose and amylopectin, yielding glucose,gamma-amylase will cleave α(1-6) glycosidic linkages.

A fourth enzyme, alpha-glucosidase, acts on maltose and other shortmalto-oligosaccharides produced by alpha-, beta-, and gamma-amylases,converting them to glucose.

Three major types of enzymatic activities are required for nativecellulose degradation: The first type are endoglucanases (1,4-β-D-glucan4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in thecellulose polysaccharide chain of amorphous cellulose, generatingoligosaccharides of varying lengths and consequently new chain ends. Thesecond type are exoglucanases, including cellodextrinases(1,4-β-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases(1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in aprocessive manner on the reducing or non-reducing ends of cellulosepolysaccharide chains, liberating either glucose (glucanohydrolases) orcellobiose (cellobiohydrolase) as major products. Exoglucanases can alsoact on microcrystalline cellulose, presumably peeling cellulose chainsfrom the microcrystalline structure. The third type are β-glucosidases(β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyzesoluble cellodextrins and cellobiose to glucose units.

A variety of plant biomass resources are available as starch andlignocellulosics for the production of biofuels, notably bioethanol. Themajor sources are (i) wood residues from paper mills, sawmills andfurniture manufacturing, (ii) municipal solid wastes, (iii) agriculturalresidues and (iv) energy crops such as corn. Pre-conversion ofparticularly the cellulosic fraction in these biomass resources (usingeither physical, chemical or enzymatic processes) to fermentable sugars(glucose, cellobiose, maltose, alpha- and cellodextrins) would enabletheir fermentation to bioethanol, provided the necessary fermentativemicro-organism with the ability to utilize these sugars is used.

On a world-wide basis, 1.3×10¹⁰ metric tons (dry weight) of terrestrialplants are produced annually (Demain, A. L., et al., Microbiol. Mol.Biol. Rev. 69, 124-154 (2005)). Plant biomass consists of about 40-55%cellulose, 25-50% hemicellulose and 10-40% lignin, depending whether thesource is hardwood, softwood, or grasses (Sun, Y. and Cheng, J.,Bioresource Technol. 83, 1-11 (2002)). The major polysaccharide presentis water-insoluble, cellulose that contains the major fraction offermentable sugars (glucose, cellobiose or cellodextrins).

Bakers' yeast (Saccharomyces cerevisiae) remains the preferredmicro-organism for the production of ethanol (Hahn-Hágerdal, B., et al.,Adv. Biochem. Eng. Biotechnol. 73, 53-84 (2001)). Attributes in favor ofthis microbe are (i) high productivity at close to theoretical yields(0.51 g ethanol produced/g glucose used), (ii) high osmo- and ethanoltolerance, (iii) natural robustness in industrial processes, (iv) beinggenerally regarded as safe (GRAS) due to its long association with wineand bread making, and beer brewing. Furthermore, S. cerevisiae exhibitstolerance to inhibitors commonly found in hydrolyzaties resulting frombiomass pretreatment. The major shortcoming of S. cerevisiae is itsinability to utilize complex polysaccharides such as starch andcellulose, or its break-down products, such as cellobiose andcellodextrins.

Genes encoding cellobiohydrolases in T. reseei (CBH1 and CBH2), A. niger(CBHA and CBHB) and P. chrysosporium (CBH1-4) have been cloned anddescribed. The proteins encoded by these genes are all modular enzymescontaining a catalytic domain linked via a flexible liner sequence to acellulose-binding molecule. CBH2 and CBHB are family 6 glycosylhydrolases. CBH1 and CBH1-4 are family 7 glycosyl hydrolases. Glycosylhydrolases are a widespread group of enzymes that hydrolyze theglycosidic bond between two or more carbohydrates, or between acarbohydrate and a non-carbohydrate moiety. A classification system forglycosyl hydrolases, based on sequence similarity, has led to thedefinition of 85 different families (Henrissat, B. et al., Proc. Natl.Acad. Sci. 92:7090-7094 (1995); Davies, G. and Henrissat, B., Structure3: 853-859 (1995)). Glycoside hydrolase family 7 (GHF7) comprisesenzymes with several known activities including endoglucanase andcellobiohydrolase. These enzymes were formerly known as cellulase familyC.

Cellobiohydrolases play a role in the conversion of cellulose to glucoseby cutting the dissaccharide cellobiose from the reducing (CBH1; GHF7)or nonreducing (CBH2; GHF6) end of the cellulose polymer chain.Structurally, cellulases and xylanases generally consist of a catalyticdomain joined to a cellulose-binding domain (CBD) via a linker regionthat is rich in proline and/or hydroxy-amino acids. In type Iexoglucanases, the CBD domain is found at the C-terminal extremity ofthese enzyme (this short domain forms a hairpin loop structurestabilised by 2 disulphide bridges). Some cellulases have only thecatalytic domain.

Glycosyl hydrolase family 7 enzymes have a 67% homology at the aminoacid level, but the homology between any of these enzymes and theglycosyl hydrolase family 6 CBH2 is less than 15%.

With the aid of recombinant DNA technology, several of theseheterologous cellulases from bacterial and fungal sources have beentransferred to S. cerevisiae, enabling the degradation of cellulosicderivatives (Van Rensburg, P., et al., Yeast 14, 67-76 (1998)), orgrowth on cellobiose (Van Rooyen, R., et al., J. Biotech. 120, 284-295(2005)); McBride, J. E., et al., Enzyme Microb. Techol. 37, 93-101(2005)).

Related work was described by Fujita, Y., et al., (Appl. Environ.Microbiol. 70, 1207-1212 (2004)) where cellulases immobilised on theyeast cell surface had significant limitations. Firstly, Fujita et al.were unable to achieve fermentation of amorphous cellulose using yeastexpressing only recombinant BGL1 and EGII. A second limitation of theFujita et al. approach was that cells had to be pre-grown to high celldensity on standard carbon sources before the cells were useful forethanol production using amorphous cellulose (e.g., Fujita et al.teaches high biomass loadings of ˜15 g/L to accomplish ethanolproduction).

As noted above, ethanol producing yeast such as S. cerevisiae requireaddition of external cellulases when cultivated on cellulosic substratessuch as pre-treated wood because this yeast does not produce endogenouscellulases. Functional expression of fungal cellulases such as T. reeseiCBH1 and CBH2 in yeast S. cerevisiae have been demonstrated (Den Haan Ret al., Metab Eng., 9, 87-94 (2007)). However, current levels ofexpression and specific activity of cellulases heterologously expressedin yeast are still not maximally efficient with respect to thelignocellulosic substrate. Thus, there remains a significant need forimprovement in the amount and variety of cellulase activity expressed inorder to attain the goal of achieving a consolidated bioprocessing (CBP)system capable of efficiently and cost-effectively converting cellulosicsubstrates to ethanol.

The composition of lignocellulosic material varies greatly based on itsspecies of origin, the particular tissue from which it is derived, andits pretreatment. Because of its varied composition, organisms designedfor CBP must produce digestive enzymes that can accommodate a variety ofsubstrates, in a variety of conformations, in a variety of reactionenvironments. To date, efficient usage of lignocellulosic substratesrequires the addition of external enzymes at high levels and externallyadded enzymes are costly. Therefore it would be very beneficial toisolate cellulases from cellulolytic organisms with high specificactivity and high expression levels in host organisms, such as the yeastS. cerevisiae in order to achieve CBP. Also, in order to uselignocellulosic material with maximal efficiency, it would also bebeneficial to discover combinations of paralogous and/or orthologousenzymes that work synergistically to achieve more efficient break downof lignocellulosic components.

The secretome of Trichoderma reesei consists of 22 unique identifiableprotein species (Herpoél-Gimbert I, Margeot A, Dolla A, et al.,Comparative secretome analyses of two Trichoderma reesei RUT-C30 andCL847 hypersecretory strains, Biotechnol Biofuels. 2008 Dec. 23;1(1):18), identified by 2D gel electrophoresis and MALDI-TOF massspectrometry. However, a study of the complementation of the T. reeseisystem, showed that the addition of a small amount of supernatant fromother cellulolytic fungi provided a substantial increase in activity forT. reesei cellulase preparations (Rosgaard L, Pedersen S, Cherry J R, etal., Efficiency of new fungal cellulase systems in boosting enzymaticdegradation of barley straw lignocellulose, Biotechnol Prog. 2006March-April; 22(2):493-8). In addition to this, a comparison of the T.reesei genome to several other cellulolytic fungi (Martinez D, Berka RM, Henrissat B, et al., Genome sequencing and analysis of thebiomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina),Nat Biotechnol. 2008 May; 26(5):553-60) found that its genome encodesfewer cellulases and hemicellulases than all of the other sequencedcellulolytic fungi, and may be particularly deficient in hemicellulosedegradation since it is missing the tannase and feruoyl esterase enzymefamilies completely. These studies suggest that activities not presentin the T. reesei genome may also be useful for hydrolyzinglignocellulose.

In addition, literature on reconstituted cellulase systems from fungi doprovide some insight into which enzymes (and how much) are needed forhydrolysis. Gusakov A V, Salanovich T N, Antonov A I, et al., Design ofhighly efficient cellulase mixtures for enzymatic hydrolysis ofcellulose, Biotechnol Bioeng. 2007 August 1; 97(5):1028-38 used purifiedChrysosporium lucknowense cellulases, and showed that a mixture of CBH1,CBH2, EG2, EG5, BGL, and XYN2 could extensively hydrolyze Organosolvpretreated douglas fir. Because the Organosolv pretreatment extensivelyremoves lignin, it is likely it would remove the need for some enzymeactivities in addition. In another study (Zhou J, Wang Y H, Chu J, etal., Optimization of cellulase mixture for efficient hydrolysis ofsteam-exploded corn stover by statistically designed experiments,Bioresour Technol. 2009 January; 100(2):819-25. Epub 2008 Sep. 3), ˜80%of the glucan in pretreated corn stover could be converted by a mix of 7enzymes, including CBH1, CBH2, EG1, EG3, EG4, and BGL. In the optimizedmix created by the authors, the CBHs made up about two-thirds of thetotal cellulase, and the ratio of CBH2 to CBH1 was 2:1. In both of thesestudies, the reconstituted systems showed greater total hydrolysis thanthe crude enzyme preparation, although this is likely a function of thepretreatment conditions.

Beyond fungi, there are a large variety of cellulolytic bacteria thatcan be used as gene donors for expression of lignocellulolytic enzymesin yeast. In one aspect, the present invention is drawn to identifyingcellulolytic enzymes from a variety of organisms and subsequentlyidentifying enzymes that work in maximally efficient combinations todigest lignocellolosic material. Given the diversity of cellulolyticbacteria, classification of these organisms based on several parameters(Lynd et al., 2002) may inform the choice of gene donors. The followingare possible distinguishing characteristics: A) aerobic vs. anaerobic,B) mesophiles vs. thermophiles; and, C) noncomplexed, cell free enzymesvs. complexed, cell bound enzymes.

Another consideration when defining the needed set of enzymaticactivities is to attempt to characterize the linkages in alignocellulosic substrate. The following is an analysis for a hardwoodsubstrate. FIG. 1 provides an overview of the carbohydrate structurespresent in plant material given in Van Zyl W H et al., Consolidatedbioprocessing for bioethanol production using Saccharomyces cerevisiae,Adv Biochem Eng Biotechnol., 108, 205-235 (2007). Although thisdepiction is not specific to hardwoods, it corresponds relatively wellwith information from the Handbook of Wood Chemistry and Composites(Rowell, 2005), which states that hardwood hemicelluloses have thefollowing characteristics: Largely comprised of glucuronoxylans—similarto structure (B) from FIG. 1. These have a xylan backbone (beta 1-4linked xylopyranose units) with acetyl groups at C2 or C-3, average of 7acetyls per ten xylose units, and are substituted with sidechains of4-O-methylglucuronic acid (alpha 1-2 linkage). Hardwoods contain 2-5% ofa glucomannan composed of beta-D-glucopyranose and beta-D-mannopyranoseunits linked 1-4—somewhat similar to structure (C) from FIG. 1; andhardwoods contain small amounts of pectins, starch and proteins.

Panel F from FIG. 1 gives the structure for a type of xylan—ligninlinkage, as well as the 4-O-methylglucuronic acid linkage to xylan thatare associated with hardwoods. This figure was taken from Spanikova Sand Biely P, FEBS Lett., 580, 4597-4601 (2006). The authors of thispaper identified an enzyme, glucuronoyl esterase, which acts on theselinkages. They identified the T. reesei Cip2 as a homologue of thisenzyme.

In order to address the limitations of heterologous cellulase expressionin consolidated bioprocessing systems, in one aspect, the presentinvention provides for the identification of novel saccharolytic enzymesthat are capable of facilitating efficient cellulase digestion andfermentation product production in host cells. In particular, in oneembodiment, the present invention is directed to the isolation of novelgenes for saccarolytic enzymes from cellulolytic organisms. The presentinvention provides novel genes that are capable of being heterologouslyexpressed in yeast systems and facilitate the digestion of starch,pentose sugars, and lignocellulosic components. Specifically, thepresent invention provides in one embodiment for novel genes forsaccharolytic enzymes from a variety of bacterial, fungal,non-conventional yeast, and plant organisms which can be expressed inyeast.

In another aspect, the present invention also describes industrial yeaststrains that express enzymes for the production of fuel ethanol fromcorn starch.

Even though yeast strains expressing enzymes for the production of fuelethanol from whole grain or starch have been previously disclosed, theapplication has not been commercialized in the grain-based fuel ethanolindustry, due to the relatively poor ability of the resulting strains toproduce/tolerate high levels of ethanol. For example, U.S. Pat. No.7,226,776 discloses that a polysaccharase enzyme expressing ethanologencan make ethanol directly from carbohydrate polymers, but the maximalethanol titer demonstrated is 3.9 g/l. U.S. Pat. No. 5,422,267 disclosesthe use of a glucoamylase in yeast for production of alcoholicbeverages; however, no commercially relevant titers of ethanol aredisclosed.

Additionally, although yeast cells are known to naturally utilize sugarssuch as glucose and mannose, they lack the ability to efficientlyutilize pentose sugars such as xylose and arabinose.

Therefore, in one embodiment, the present invention describes industrialyeast strains that are engineered to express a broad spectrum of varioussaccharolytic enzymes as well as pentose utilization pathways forproduction of various compounds from biomass feedstock containing mix ofhexose and pentose mono- and poly-saccharides.

Engineering and utilization of such yeast strain(s) would allow abioprocess with a biomass feedstock. Such biomass feedstock couldinclude several different polymeric compounds such as: cellulose,hemicellulose, starch, pectin, inulin, levan and others. Also, thebiomass feedstock could contain the mix of pentose and hexosecarbohydrates. Therefore, complex substrates derived from plants such aswood, corn, agave, switch grass and others that contain combination ofdifferent carbohydrates and carbohydrate polymers could be utilized in abioprocess without prior separation of different substrates.Furthermore, substrates derived from different sources could be combinedin the same bioprocess. The substrates could be derived directly fromplants or from any kind of waste or byproducts containing carbohydrates.

The present invention represents the first demonstration of a full CBPeffect at commercial ethanol production level, wherein yeast producedenzymes completely replace exogenous enzyme added in standard commercialprocess. As a result, a yeast CBP strain was able to produce over 125g/l ethanol from liquefied corn mash in 72 hrs without any exogenousenzymes added. This was achieved due to engineering selected set ofenzymes into an industrial robust background strain. The resultingstrains may also be used to produce ethanol directly from granularstarch without liquefaction.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, the invention comprising a yeast strain, orstrains, secreting a full suite, or a subset of that full suite, ofenzymes to hydrolyze lignocellulose, including enzymes that hydrolyzechemical linkages in cellulose, hemicellulose, and between lignin andcarbohydrates. In some embodiments, the invention is also a set ofproteins that are well-expressed in yeast for each category of necessaryenzymatic activity in order to efficiently utilize a particularlignocellulosic material. This full suite of enzymes contains activitiesbeyond those identified previously for expression in yeast: CBH1, CBH2,EG, and BGL (as disclosed e.g. in PCT Application No.PCT/US2009/065571). In some embodiments, the present invention relatesto a yeast cell that expresses one or more gene products of the genes:Aspergillus fumigatus Endoglucanase (Accession No. XP_747897);Neosartorya fischeri Endoglucanase (Accession No. XP_001257357);Aspergillus clavatus Endoglucanase (Accession No. XP_001270378);Aspergillus terreus Endoglucanase (Accession No. XP_001217291);Penicillium marneffei Endoglucanase (Accession No. XP_002152969);Chaetomium globosum Endoglucanase (Accession No. XP_001229968);Neurospora crassa Endoglucanase (Accession No. XP_956431); Aspergillusoryzae Endoglucanase (Accession No. BAA22589); Thielavia heterothallicaEndoglucanase (Accession No. AAE25067); Fusarium oxysporum Endoglucanase(Accession No. AAG09047); Humicola insolens Endoglucanase (Accession No.1DYM_A); Pyrenophora tritici-repentis Endoglucanase (Accession No.XP_001935476); Magnaporthe grisea Endoglucanase (Accession No.XP_370166); Fusarium graminearum Endoglucanase (Accession No.XP_388429); Chrysosporium lucknowense Endoglucanase; Polyporusarcularius Endoglucanase (Accession No. BAF75943.1); Aspergilluskawachii Endoglucanase (Accession No. BAB62317.1); Heterodera schachtiiEndoglucanase (Accession No. CAC12958.1); Orpinomyces sp. Endoglucanase(Accession No. AAD04193.1); Irpex lacteus Endoglucanase (Accession No.BAD67544.1); Chaetomium globosum Endoglucanase (Accession No.XP_001220409.1); Aspergillus niger Endoglucanase (Accession No.XP001397982.1); Penicillium decumbens Endoglucanase (Accession No.ABY28340.1); Phanerochaete chrysosporium Endoglucanase (Accession No.AAU12276); Stachybotrys echinata Endoglucanase (Accession No. AAM77710);Neosartorya fischeri Endoglucanase (Accession No. XP001261563);Chaetomium brasiliense Endoglucanase (Accession No. AAM77701);Chaetomium globosum Endoglucanase (Accession No. EAQ86340); Aspergillusfumigatus Endoglucanase (Accession No. CAF31975); Humicola insolensEndoglucanase (Accession No. CAG27577); Neosartorya fischeriEndoglucanase (Accession No. XP001267517); Thielavia terrestrisEndoglucanase (Accession No. ACE10231); Chrysosporium lucknowenseEndoglucanase (Accession No. ACH15008); Chaetomium globosumEndoglucanase (Accession No. XP_001226436); Acremonium thermophilumEndoglucanase (Accession No. ACE10216); Humicola insolens Endoglucanase(Accession No. CAB42307); Thielavia terrestris Endoglucanase (AccessionNo. CAH03187); Chrysosporium lucknowense Endoglucanase (Accession No.AAQ38151); Magnaporthe grisea Endoglucanase (Accession No. EDJ97375);Chaetomium globosum Endoglucanase (Accession No. EAQ84577); Humicolainsolens Endoglucanase 1DYS_B; Neurospera crassa Endoglucanase(Accession No. XP_957415); Trichoderma reesei Xyloglucanase (AccessionNo. AAP57752); Aspergillus niger Xyloglucanase (Accession No. AAK77227);Aspergillus aculeatus Xyloglucanase (Accession No. BAA29031);Neosartorya fischeri Xyloglucanase (Accession No. XP_001261776);Chaetomium thermophilum Endoxylanase (Accession No. CAD48749);Trichoderma reesei Endoxylanase (Accession No. ABK59833); Chrysosporiumlucknowense Endoxylanase (Accession No. AAQ38147); Aureobasidiumpullulans Endoxylanase (Accession No. BAE71410); Aspergillus nidulansbeta-xylosidase (Accession No. CAA73902; Cochliobolus carbonumbeta-xylosidase (Accession No. AAC67554); Penicillium herqueibeta-xylosidase (Accession No. BAC75546); Pyrenophora tritici-repentisbeta-xylosidase (Accession No. XP_001940956); Aspergillus nigerbeta-mannosidase (Accession No. Q9UUZ3); Aspergillus aculeatusbeta-mannosidase (Accession No. BAA29029); Neosartorya fischeribeta-mannosidase (Accession No. XP_001258000); Trichoderma reeseialpha-glucuronidase (Accession No. CAA92949); Aspergillus nigeralpha-glucuronidase (Accession No. CAC38119); Talaromyces emersoniialpha-glucuronidase (Accession No. AAL33576); Aspergillus nigeracetylxylanesterase (Accession No. XP_001395572); Trichoderma reeseiacetylxylanesterase (Accession No. Q99034); Neosartorya fischeriacetylxylanesterase (Accession No. XP_001262186); Trichoderma reeseiarabinofuranosidase, 1,4-beta-D-arabinoxylan arabinofuranohydrolase(Accession No. AAP57750); Chaetomium globosum arabinofuranosidase,1,4-beta-D-arabinoxylan arabinofuranohydrolase (Accession No.XP001223478); Aspergillus niger arabinofuranosidase,1,4-beta-D-arabinoxylan arabinofuranohydrolase (Accession No.XP_001389998); Penicillium decumbens Swollenin (Accession No. ACH57439);Neosartorya fischeri Swollenin (Accession No. XP001257521); Talaromycesstipitatus Swollenin (Accession No EED19018); Trichoderma reesei(Accession No. AAP57751); Chaetomium globosum (Accession No.XP001228455); Magnaporthe grisea (Accession No. XP_365869); Trichodermareesei glucuronyl esterase (Accession No. AAP57749); Chaetomium globosumglucuronyl esterase (Accession No. XP_001226041); Aspergillus fumigatusglucuronyl esterase (Accession No. XP_751313); Populus albaalpha-expansin (Accession No. BAB39482); Vitis lubrusca alpha-expansin(Accession No. BAC66697); Triticum aestivum beta-expansin (Accession No.AAS48881); Eucalyptus globulus beta-expansin (Accession No. AAZ08315);Aspergillus niger Feruoyl esterase (Accession No. XP001393337);Aspergillus terreus Feruoyl esterase (Accession No. XP001211092);Talaromyces stipitatus Feruoyl esterase (Accession No. EED17739);Chaetomium globosum Feruoyl esterase (Accession No. XP_001228412)Streptomyces avermitilis 1,4-beta-cellobiosidase guxA1 (Accession No.NP_821732.1); Streptomyces avermitilis 1,4-beta-cellobiosidase guxA2(Accession No. NP_823029.1); Streptomyces avermitilis1,4-beta-cellobiosidase guxA3 (Accession No. NP_823031.1); Streptomycesavermitilis Endo-1,4-beta-glucanase celA1 (Accession No. NP_821730.1);Streptomyces avermitilis Endo-1,4-beta-glucanase celA2 (Accession No.NP_823030.1); Streptomyces avermitilis Endo-1,4-beta-glucanase celA3(Accession No. NP_823032.1); Streptomyces avermitilisEndo-1,4-beta-glucanase celA4 (Accession No. NP_823744.1); Streptomycesavermitilis Endo-1,4-beta-glucanase (Accession No. NP_826394.1);Streptomyces avermitilis Endo-1,4-beta-glucanase celA5 (Accession No.NP_828072.1); Streptomyces avermitilis Beta-1,4-xylanase (Accession No.NP_823272.1); Streptomyces avermitilis Beta-1,4-xylanase (Accession No.NP_826161.1); Streptomyces avermitilis Xylanase (Accession No.NP_827548.1); Streptomyces avermitilis Endo-1,4-beta-xylanase xynD(Accession No. NP_827557.1); Streptomyces avermitilis1,4-beta-xylosidase xynB1 (Accession No. NP_822628.1); Streptomycesavermitilis Beta-xylosidase (Accession No. NP_823285.1); Streptomycesavermitilis 1,4-beta-xylosidase xynB2 (Accession No. NP_826159.1);Streptomyces avermitilis 1,4-beta-xylosidase xynB3 (Accession No.NP_827745.1); Streptomyces avermitilis Beta-glucosidase bglC1 (AccessionNo. NP_822977.1); Streptomyces avermitilis Beta-glucosidase bglC2(Accession No. NP_826430.1); Streptomyces avermitilis Beta-glucosidasebglC3 (Accession No. NP_826775.1); Streptomyces avermitilis AXE1(Accession No. NP_822477.1); Streptomyces avermitilis AXE1 (AccessionNo. NP_822632.1); Streptomyces avermitilis abfA (Accession No.NP_822218.1); Streptomyces avermitilis abfB (Accession No. NP_822290.1);Streptomyces avermitilis abfA (Accession No. NP_826920.1); Streptomycesavermitilis abfB (Accession No. BAC74043.1); Streptomyces avermitilisSAV_6756 (Accession No. BAC74467.1); Streptomyces avermitilis agaA1(Accession No. BAC68338.1); Streptomyces avermitilis agaA3 (AccessionNo. BAC68787.1); Streptomyces avermitilis agaB2 (Accession No.BAC69185.1); Saccharophagus degradans 2-40 Sde_2993 (Accession No.YP_528462.1); Saccharophagus degradans 2-40 Sde_2996 (Accession No.YP_528465.1); Saccharophagus degradans 2-40 Sde_3023 (Accession No.YP_528492.1); Saccharophagus degradans 2-40 cel5A (Accession No.ABD82260.1); Saccharophagus degradans 2-40 cel5E (Accession No.ABD82186.1); Saccharophagus degradans 2-40 cel5F (Accession No.ABD80834.1); Saccharophagus degradans 2-40 cel5J (Accession No.ABD81754.1; Saccharophagus degradans 2-40 cel9A (Accession No.ABD79898.1); Saccharophagus degradans 2-40 ced3A (Accession No.ABD81757.1); Saccharophagus degradans 2-40 ced3B (Accession No.ABD79509.1); Saccharophagus degradans 2-40 bgl1A (Accession No.ABD82858.1); Saccharophagus degradans 2-40 bgl1B (Accession No.ABD80656.1); Saccharophagus degradans 2-40 Cep94A (Accession No.ABD80580.1); Saccharophagus degradans 2-40 Cep94B (Accession No.ABD80168.1); Saccharophagus degradans 2-40 Sde_0509 (Accession No.YP_525985.1); Saccharophagus degradans 2-40 Sde_0169 (Accession No.YP_525645.1); Bacillus subtilis Expansin ex1X (Accession No.CAB13755.1); Bacillus subtilis Endo-1,4-beta-glucanase eglS (AccessionNo. CAB13696.2); Bacillus subtilis Endo-xylanase xynC (Accession No.CAB13698.1); Bacillus subtilis Endo-1,4-beta-xylanase xynD (AccessionNo. CAB13699.1); Bacillus subtilis Endo-1,4-beta-xylanase xynA(Accession No. CAB13776.1); Bacillus subtilis Xylan beta-1,4-xylosidasexynB (Accession No. CAB13642.2); Clostridium phytofermentans Cphy_3367(Accession No. YP_001560459.1); Clostridium phytofermentans Cphy_3368(Accession No. YP_001560460.1); Clostridium phytofermentans Cphy_2058(Accession No. YP_001559165.1); Clostridium phytofermentans Cphy_3202cellulase B (Accession No. YP_001560295.1); Clostridium phytofermentansCphy_1163 (Accession No. YP_001558280.1); Clostridium phytofermentansCphy_3329 (Accession No. YP_001560421.1); Clostridium phytofermentansCphy_1125 (Accession No. YP_001558242.1); Clostridium phytofermentansCphy_1510 (Accession No. YP_001558623.1); Clostridium phytofermentansCphy_0624 (Accession No. YP_001557750.1); Clostridium phytofermentansCphy_2105 XynA (Accession No. YP_001559210.1); Clostridiumphytofermentans Cphy_2108 (Accession No. YP_001559213.1); Clostridiumphytofermentans Cphy_3207 Y (Accession No. YP_001560300.1); Clostridiumphytofermentans Cphy_0191 (Accession No. YP_001557317.1); Clostridiumphytofermentans Cphy_0875 (Accession No. YP_001558000.1); Clostridiumphytofermentans Cphy_1169 (Accession No. YP_001558286.1); Clostridiumphytofermentans Cphy_1071 (Accession No. YP_001558190.1); Clostridiumphytofermentans Cphy_2128 (Accession No. YP_001559233.1); Clostridiumphytofermentans Cphy_2276 (Accession No. YP_001559376.1); Clostridiumphytofermentans Cphy_1936 (Accession No. YP_001559043.1); Clostridiumcellulolyticum cel5I (Accession No. AAL79562.1); Clostridiumcellulolyticum CelCCF (dockerin) Cel48F-yeast CO template pMU914(Accession No. AAB41452.1); Clostridium cellulolyticum Ccel_1259(Accession No. YP_002505595); Clostridium cellulolyticum Ccel_2226(Accession No. YP_002506548.1); Clostridium cellulolyticum Ccel_0732(dockerin) Cel9E-yeast CO template pMU913 (Accession No.YP_002505091.1); Clostridium cellulolyticum Ccel_1099 (dockerin)Cel5A-yeast CO template pMU967 (Accession No. YP_002505438.1);Clostridium cellulolyticum Ccel_2392 (dockerin) (Accession No.YP_002506705.1); Clostridium cellulolyticum Ccel_0731 (dockerin)Cel9G-yeast CO template pMU892 (Accession No. YP_002505090.1);Clostridium cellulolyticum Ccel_0840 (dockerin) Cel5D-yeast CO templatepMU891 (Accession No. YP_002505196.1); Clostridium cellulolyticum CelCCC(dockerin) Cel8C-yeast CO template pMU969 (Accession No. AAA73867.1);Thermobifida fusca endo-1,4-beta xylanase (Accession No. ABL73883.1);Thermobifida fusca endo-1,4-beta-D-xylanase (xyl11) (Accession No.AAV64879.1); Thermobifida fusca Endoglucanase (Accession No.AAZ55112.1); Thermobifida fusca cellulase (Accession No. AAZ56745.1);Thermobifida fusca exo-1,4-beta-glucosidase (Accession No. AAZ55642.1);Thermobifida fusca beta-glucosidase (Accession No. AAZ55664.1);Thermobifida fusca cellulose 1,4-beta-cellobiosidase (Accession No.YP_290015.1); Thermobifida fusca CBD E8 (Accession No. AAZ55700.1);Thermobifida fusca celC (E3) (Accession No. YP_288681.1); Thermobifidafusca celE (E5) (Accession No. YP_288962.1); Thermobifida fusca cel5B(Endoglucanase) (Accession No. AAP56348.1); Thermobifida fusca celA (E1)(Accession No. AAC06387.1); Thermobifida fusca celB (E2) (Accession No.YP_289135.1); Thermobifida fusca Tfu_1627 (1,4-beta-cellobiosidase)(Accession No. YP_289685.1); Clostridium thermocellum celA (dockerin)(Accession No. YP_001036701.1); Clostridium thermocellum celY (cel48Y)(Accession No. CAI06105.1); Clostridium thermocellum Cthe_0625(dockerin) (Accession No. YP_001037053.1); Clostridium thermocellum celC(Accession No. CAC27410.1); Clostridium thermocellum (Accession No.YP_001037893.1); Clostridium thermocellum (Accession No.YP_001038519.1); Clostridium thermocellum bglA (Accession No.CAA42814.1); Clostridium thermocellum bglB (Accession No. CAA33665.1);Clostridium thermocellum Cthe_2548 (Accession No. YP_001038942.1);Clostridium thermocellum Cthe_1273 (Accession No. YP_001037698.1);Clostridium thermocellum Cthe_0040 (Cel9I) (Accession No.YP_001036474.1); Clostridium thermocellum Cthe_0412 (dockerin)(Accession No. YP_001036843.1); Clostridium thermocellum Cthe_0825(dockerin) (Accession No. YP_001037253.1); Clostridium stercorarium xynA(Accession No. CAD48307); Clostridium stercorarium xynB(CelW—celloxylanase) (Accession No. CAD48313); Clostridium stercorariumxynC (CelX—celloxylanase) (Accession No. CAD48314); Clostridiumstercorarium bxlB (b-Xylosidase B) (Accession No. AJ508405); Clostridiumstercorarium bxlA (b-Xylosidase A) (Accession No. AJ508404); Clostridiumstercorarium bglZ (beta-glucosidase) (Accession No. CAB08072);Clostridium stercorarium arfA (alpha-arabinofuranosidaseA) (AccessionNo. AJ508406); Clostridium stercorarium arfB(alpha-arabinofuranosidaseB) (Accession No. AAC28125); Clostridiumstercorarium celZ (Cs-Cel9Z—Avicellase I) (Accession No CAA39010);Clostridium stercorarium celY (Cs-Cel48Y—Avicellase II) (Accession No.CAA93280); Anaerocellum thermophilum celA (1,4-beta-glucanase)(Accession No. CAB06786); Anaerocellum thermophilum celD (EG) (AccessionNo. CAB01405); Anaerocellum thermophilum xynA (1,4-beta-D-xylanxylanhydrolase) (Accession No. CAA93627); Anaerocellum thermophilum celB(EG5) (Accession No. Z86104); Anaerocellum thermophilum Athe_1866(endo-1,4-beta-mannosidase) (Accession No. YP_002573059); Anaerocellumthermophilum Athe_0594 (“cellulase”) (Accession No. YP_002572493).

In some embodiments, the cells of the invention can express pairs ofenzymes that have synergistic activity with respect to their action on agiven lignocellulosic substrate. Such pairs include, but are not limitedto (Streptomyces avermitilis endo-1,4-beta-glucanase celA2 (AccessionNo. NP_823030.1) and Streptomyces avermitilis endo-1,4-beta-glucanasecelA5 (Accession No. NP_828072.1)); (Streptomyces avermitilisendo-1,4-beta-glucanase celA2 (Accession No. NP_823030.1) and Bacillussubtilis endo-1,4-beta-glucanase (Accession No CAB13696.2));(Streptomyces avermitilis endo-1,4-beta-glucanase celA3 (Accession No.NP_823032.1) and Streptomyces avermitilis endo-1,4-beta-glucanase(Accession No. NP_826394.1)); (Streptomyces avermitilisendo-1,4-beta-glucanase celA4 (Accession No. NP_823744.1) andStreptomyces avermitilis xylanase (Accession No. NP_827548.1));(Bacillus subtilis endo-1,4-beta-glucanase (Accession No CAB13696.2) andStreptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_826394.1)); (Streptomyces avermitilis endo-1,4-beta-glucanase celA4(Accession No. NP_823744.1) and Bacillus subtilisendo-1,4-beta-glucanase (Accession No CAB13696.2)); (Streptomycesavermitilis endo-1,4-beta-glucanase celA5 (Accession No. NP_828072.1)and Streptomyces avermitilis endo-1,4-beta-glucanase celA4 (AccessionNo. NP_823744.1)); (Streptomyces avermitilis endo-1,4-beta-glucanasecelA5 (Accession No. NP_828072.1) and Clostridium phytofermentansxylanase (Accession No. YP_001557750.1)); (Saccharophagus degradans 2-40mannanase (Accession No. YP_525985.1) and Streptomyces avermitilisendo-1,4-beta-glucanase (Accession No. NP_826394.1)); (Streptomycesavermitilis xylanase (Accession No. NP_827548.1) and Saccharophagusdegradans 2-40 mannanase (Accession No. YP_525985.1)); (Clostridiumphytofermentans xylanase (Accession No. YP_001557750.1) and Streptomycesavermitilis xylanase (Accession No. NP_827548.1)); (Clostridiumphytofermentans xylanase (Accession No. YP_001557750.1) and Streptomycesavermitilis xylanase (Accession No. NP_827548.1)); (Streptomycesavermitilis endo-1,4-beta-glucanase celA5 (Accession No. NP_828072.1)and Streptomyces avermitilis xylanase (Accession No. NP_827548.1));(Streptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_823744.1) and Saccharophagus degradans 2-40 mannanase (Accession No.YP_525985.1)); (Streptomyces avermitilis endo-1,4-beta-glucanase celA2(Accession No. NP_823030.1) and Saccharophagus degradans 2-40 mannanase(Accession No. YP_525985.1)); (Streptomyces avermitilisendo-1,4-beta-glucanase (Accession No. NP_823744.1) and Streptomycesavermitilis endo-1,4-beta-glucanase celA3 (Accession No. NP_823032.1));(Streptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_823744.1) and Clostridium phytofermentans xylanase (Accession No.YP_001557750.1)); (Streptomyces avermitilis xylanase (Accession No.NP_827548.1) and Streptomyces avermitilis endo-1,4-beta-glucanase celA3(Accession No. NP_823032.1)); (Streptomyces avermitilisendo-1,4-beta-glucanase celA4 (Accession No. NP_823744.1) andStreptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_826394.1))

In some embodiments, host cells of the invention can express threeenzymes that have synergistic activity with respect to their action on agiven lignocellulosic substrate. Such triplets of enzymes can be, forexample (Streptomyces avermitilis endo-1,4-beta-glucanase celA4NP_823744.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA5NP_828072.1, and Streptomyces avermitilis endo-1,4-beta-glucanase celA2NP_823030.1); (Streptomyces avermitilis xylanase NP_827548.1,Streptomyces avermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, andStreptomyces avermitilis endo-1,4-beta-glucanase celA2 NP_823030.1);(Clostridium phytofermentans xylanase YP_001557750.1, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomycesavermitilis endo-1,4-beta-glucanase celA2 NP_823030.1); (Saccharophagusdegradans 2-40 mannanase YP_525985.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1); (Streptomyces avermitilisendo-1,4-beta-glucanase celA3 NP_823032.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1); (Bacillus subtilisendo-1,4-beta-glucanase eglS CAB13696.2, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1); (Streptomyces avermitilisendo-1,4-beta-glucanase NP_826394.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1); (Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA4 NP_823744.1); (Streptomyces avermitilisxylanase NP_827548.1 Streptomyces avermitilis endo-1,4-beta-glucanasecelA5 NP_828072.1, and Streptomyces avermitilis endo-1,4-beta-glucanasecelA4 NP_823744.1); (Clostridium phytofermentans xylanaseYP_001557750.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA5NP_828072.1, and Streptomyces avermitilis endo-1,4-beta-glucanase celA4NP_823744.1); (Saccharophagus degradans 2-40 mannanase YP_525985.1,Streptomyces avermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, andStreptomyces avermitilis endo-1,4-beta-glucanase celA4 NP_823744.1);(Streptomyces avermitilis endo-1,4-beta-glucanase celA3 NP_823032.1,Streptomyces avermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, andStreptomyces avermitilis endo-1,4-beta-glucanase celA4 NP_823744.1);(Streptomyces avermitilis endo-1,4-beta-glucanase NP_826394.1,Streptomyces avermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, andStreptomyces avermitilis endo-1,4-beta-glucanase celA4 NP_823744.1);(Bacillus subtilis endo-1,4-beta-glucanase eglS CAB13696.2, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomycesavermitilis endo-1,4-beta-glucanase celA4 NP_823744.1); (Streptomycesavermitilis endo-1,4-beta-glucanase celA2 NP_823030.1, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomycesavermitilis xylanase NP_827548.1); (Streptomyces avermitilisendo-1,4-beta-glucanase celA4 NP_823744.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisxylanase NP_827548.1); (Clostridium phytofermentans xylanaseYP_001557750.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA5NP_828072.1, and Streptomyces avermitilis xylanase NP_827548.1);(Saccharophagus degradans 2-40 mannanase YP_525985.1, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomycesavermitilis xylanase NP_827548.1); (Streptomyces avermitilisendo-1,4-beta-glucanase celA3 NP_823032.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisxylanase NP_827548.1); (Streptomyces avermitilis endo-1,4-beta-glucanaseNP_826394.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA5NP_828072.1, and Streptomyces avermitilis xylanase NP_827548.1);(Bacillus subtilis endo-1,4-beta-glucanase eglS CAB13696.2, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomycesavermitilis xylanase NP_827548.1); (Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Clostridiumphytofermentans xylanase YP_001557750.1); (Streptomyces avermitilisendo-1,4-beta-glucanase celA4 NP_823744.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Clostridiumphytofermentans xylanase YP_001557750.1); (Streptomyces avermitilisxylanase NP_827548.1, Streptomyces avermitilis endo-1,4-beta-glucanasecelA5 NP_828072.1, and Clostridium phytofermentans xylanaseYP_001557750.1); (Saccharophagus degradans 2-40 mannanase YP_525985.1,Streptomyces avermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, andClostridium phytofermentans xylanase YP_001557750.1); (Streptomycesavermitilis endo-1,4-beta-glucanase celA3 NP_823032.1, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Clostridiumphytofermentans xylanase YP_001557750.1); (Streptomyces avermitilisendo-1,4-beta-glucanase NP_826394.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Clostridiumphytofermentans xylanase YP_001557750.1); and, (Bacillus subtilisendo-1,4-beta-glucanase eglS CAB13696.2, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Clostridiumphytofermentans xylanase YP_001557750.1)

In some embodiments, host cells of the invention can express fourenzymes that have synergistic activity with respect to their action on agiven lignocellulosic substrate. Such quadruplets of enzymes can be, forexample (Streptomyces avermitilis endo-1,4-beta-glucanase celA4NP_823744.1, Streptomyces avermitilis xylanase NP_827548.1, Streptomycesavermitilis endo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomycesavermitilis endo-1,4-beta-glucanase celA2 NP_823030.1); (Clostridiumphytofermentans xylanase YP_001557750.1, Streptomyces avermitilisxylanase NP_827548.1, Streptomyces avermitilis endo-1,4-beta-glucanasecelA5 NP_828072.1, and Streptomyces avermitilis endo-1,4-beta-glucanasecelA2 NP_823030.1); (Clostridium phytofermentans xylanaseYP_001557750.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA4NP_823744.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA5NP_828072.1, and Streptomyces avermitilis endo-1,4-beta-glucanase celA2NP_823030.1); (Streptomyces avermitilis endo-1,4-beta-glucanaseNP_826394.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA4NP_823744.1, Streptomyces avermitilis endo-1,4-beta-glucanase celA5NP_828072.1, and Streptomyces avermitilis endo-1,4-beta-glucanase celA2NP_823030.1); (Saccharophagus degradans 2-40 mannanase YP_525985.1,Streptomyces avermitilis xylanase NP_827548.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1); and, (Saccharophagusdegradans 2-40 mannanase YP_525985.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA4, NP_823744.1, Streptomyces avermitilisendo-1,4-beta-glucanase celA5 NP_828072.1, and Streptomyces avermitilisendo-1,4-beta-glucanase celA2 NP_823030.1)

In some embodiments, the yeast cell expresses any one or more of theabove-named genes in conjunction with one or more CBH1, CBH2, EG, orBGL.

In some embodiments, the cells of the invention can be used to reducethe amount of external enzyme needed to hydrolyze lignocellulose duringan SSF or CBP process, or to increase the yield of a fermentationproduct during SSF or CBP at a given cellulase loading.

In some embodiments, the invention provides polynucleotide and aminoacid sequences of endoglucanases, xylanases, xylosidases, esterases,other hydrolases, and other accessory enzymes that are active andwell-expressed by S. cerevisiae and other yeast species. In someembodiments, these well-expressed enzymes provide an increased abilityof cellulase cocktails to hydrolyze lignocellulose. In some embodiments,combinations of the enzymes of the present invention are useful forincreasing the activity of yeast expressed “core” cellulases, CBH1,CBH2, EG, and BGL. In some embodiments, the host yeast cell expresses,in addition to the “core” cellulases, xylanase, xylosidase,glucoamylase, and acetixylan esterase. In some embodiments, theinvention provides technology for expressing multiple genes in multiplecopies using yeast high-expression vectors, centromeric vectors and bygenomic integration.

In some embodiments, the present invention relates to processes ofproducing fermentation products by contacting cells of the inventionwith lignocellulosic material and then recoving the fermentationmaterial.

In some embodiments, the invention relates to the products produced bythe fermentation of lignocellulosic materials.

In one aspect, the saccharolytic enzymes (amylases, cellulases,hemicellulases, cellulolytic and amylolytic accessory enzymes,inulinases, levanases, and others) and pentose utilizing enzymes arecombined in a single yeast strain. In another embodiment, the hydrolyticand pentose hydrolyzing enzymes are expressed in different yeast strainsused in the same technological process. In one aspect, yeast strains,each expressing a different enzyme, or a different combination ofenzymes, are co-cultured in the same volume. In another embodiment,yeast strains, each expressing a different enzyme, or a differentcombination of enzymes, are cultured in separate tanks.

Complex biomass feedstocks contain varying amounts of starch,lignocellulosic material, and pentose sugars. Accordingly, the yeaststrains of the present invention are constructed to express differentsaccharolytic enzymes at different levels. In one embodiment, a yeaststrain expresses one or more cellulolytic enzymes at a higher level thanone or more amylolytic enzymes and one or more pentose sugar utilizingenzymes. In another embodiment, the yeast strain expresses one or moreamylolytic enzymes at a higher level than one or more cellulolyticenzymes and one or more pentose sugar utilizing enzymes. In yet anotherembodiment, the yeast strain expresses one or more pentose sugarutilizing enzymes at a higher level than one or more cellulolyticenzymes and one or more amylolytic enzymes.

In some embodiments, the present invention relates to a recombinantyeast host cell comprising a heterologous polynucleotide encoding apolypeptide comprising an amino acid sequence at least 90% identical toany one of the amino acid sequences of SEQ ID NOs: 442-446.

In some embodiments, the present invention relates to a recombinantyeast host cell comprising one or more heterologous polynucleotidesencoding a polypeptide of Table 19.

In some embodiments, the present invention relates to a recombinantyeast host cell comprising: (a) at least one heterologous polynucleotidecomprising a nucleic acid which encodes a glucoamylase; (b) at least oneheterologous polynucleotide comprising a nucleic acid which encodes analpha-glucosidase; (c) at least one heterologous polynucleotidecomprising a nucleic acid which encodes an enzyme that utilizes pentosesugar; and (d) further comprising at least one heterologouspolynucleotide encoding a polypeptide comprising an amino acid sequenceaccording to SEQ ID NOs: 442-446. In another embodiment, the yeast hostcell further comprises an alpha-amylase, a pullulanse, and/or anisopullulanse.

In some embodiments, the cells of the invention can express pairs ofamylolytic enzymes that have synergistic activity with respect to theiraction on a given biomass substrate. Such pairs include, but are notlimited to (SEQ ID NO: 443 and SEQ ID NO: 444); (SEQ ID NO: 443 and SEQID NO: 445); (SEQ ID NO: 445 and SEQ ID NO: 446); (SEQ ID NO: 443 andSEQ ID NO: 445); (SEQ ID NO: 442 and SEQ ID NO: 445); (SEQ ID NO: 444and Bacillus subtilis arabinoxylanase (Accession No. CAB13699.1)); (SEQID NO: 444 and Bacillus subtilis arabinoxylanase (Accession No.CAB13699.1)); (SEQ ID NO: 444 and Bacillus subtilis arabinanendo-1,5-alpha-L-arabinosidase (Accession No. CAB15969.1)); (SEQ ID NO:444 and Bacillus subtilis arabinan-endo 1,5-alpha-L-arabinase (AccessionNo. CAA99586.1)); (SEQ ID NO: 444 and Bacillus subtilis arabinanendo-1,5-alpha-L-arabinosidase (Accession No. AL009126)); (SEQ ID NO:444 and Bacillus subtilis endo-arabinase (Accession No. D85132)); (SEQID NO: 444 and Clostridium phytofermentans arabinogalactanendo-1,4-beta-galactosidase (Accession No. CP000885)); (SEQ ID NO: 444and Bacillus licheniformis arabinan-endo 1,5-alpha-L-arabinase(Accession No. AAU40201.1); (SEQ ID NO: 444 and Bacillus licheniformisarabinan-endo 1,5-alpha-L-arabinase (Accession No. AAU41895.1); (SEQ IDNO: 444 and Bacillus licheniformis arabinogalactanendo-1,4-beta-galactosidase (Accession No. AAU43089.1); (SEQ ID NO: 444and Bacillus licheniformis arabinan endo-1,5-alpha-L-arabinosidase(Accession No. AAU43033.1); (SEQ ID NO: 444 and Bacillus licheniformisarabinan endo-1,4-beta-xylanase (Accession No. AAU39947.1); (SEQ ID NO:444 and Thermoanaerobacterium saccharolyticum arabinogalactanendo-1,4-beta-galactosidase); (SEQ ID NO: 444 and Thermoanaerobacteriumsaccharolyticum alpha-N-arabinofuranosidase); (SEQ ID NO: 444 andStreptomyces avermitilis endo-1,4-beta-xylanase xynD (Accession No.827557.1); (SEQ ID NO: 444 and Bacillus subtilis endo-1,4-beta-xylanasexynA (Accession No. CAB13776.1); (SEQ ID NO: 444 and Clostridiumphytofermentans xylanase (Accession No. YP_001558623.1); (SEQ ID NO: 444and Clostridium phytofermentans xylanase (Accession No. YP_001557750.1);(SEQ ID NO: 444 and Thermobifida fusca endo-1,4-beta-D-xylanase (xyl11)(Accession No. AAV64879.1); (SEQ ID NO: 444 and Clostridium thermocellumxylanase (Accession No. YP_001038519.1); (SEQ ID NO: 444 and Clostridiumstercorarium endo-xylanase (Accession No. CAD48307); (SEQ ID NO: 444 andClostridium stercorarium xynC (CelX—celloxylanase) (Accession No.CAD48314); (SEQ ID NO: 444 and Aspergillus niger alpha-glucosidase(Accession No. BAA23616.1)); (SEQ ID NO: 444 and Thermoanaerobacteriumsaccharolyticum glucoamylase).

In some embodiments, host cells of the invention can express threeenzymes that have synergistic activity with respect to their action on agiven biomass substrate. Such triplets of enzymes can be, for example(SEQ ID NO: 442, SEQ ID NO: 445 and SEQ ID NO: 446); (SEQ ID NO: 444,SEQ ID NO: 445 and SEQ ID NO: 446); (SEQ ID NO: 442, SEQ ID NO: 445 andSEQ ID NO: 446).

In some embodiments, host cells of the invention can express fourenzymes that have synergistic activity with respect to their action on agiven biomass substrate. Such quadruplets of enzymes can be, for example(SEQ ID NO: 442, SEQ ID NO: 444, SEQ ID NO: 445 and SEQ ID NO: 446);(SEQ ID NO: 443, SEQ ID NO: 444, SEQ ID NO: 445 and SEQ ID NO: 446).

In some embodiments, the present invention relates to a method ofproducing a fermentation product comprising: (a) combining a yeast cellof any one of claims 1-34 with grain feedstock; (b) allowing the yeastcell to ferment the grain feedstock; and (c) recovering one or moreproducts of the fermentation.

In some embodiments, the present invention relates to a recombinantyeast host cell comprising two or more heterologous polynucleotidesencoding a polypeptide comprising: (a) at least one amino acid sequencesat least 90% identical to one or more of the amino acid sequences of SEQID NOs: 219-436; and (b) at least one amino acid sequences at least 90%identical to one or more of the amino acid sequences of SEQ ID NOs:442-446.

In some embodiments, the present invention relates to a recombinantyeast host cell comprising: (a) at least one heterologous polynucleotideencoding a polypeptide of Table 11; and (b) at least one heterologouspolynucleotide encoding a polypeptide of Table 19.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the complexity of cellulose and hemicellulose and theenzymes involved in their degradation. Cellulose (a) and hemicellulosestructures for arabinoxylan (b), galactomannan (c), and xyloglucan (d)depicting the different side chains present. Hexoses are distinguishedfrom pentoses by the presence of a protruding line from the cyclichexagon (pyranose ring), depicting the CH₂OH group. Hydrolase enzymesand the bonds targeted for cleavage in the four polysaccharidestructures are indicated by arrow.

FIG. 2 depicts a basic cloning and expression vector for testingcellulases (pMU1531). This vector is an episomal 2-μ yeast expressionvector used for expression of genes in yeast. ENO1 promoter—S.cerevisiae ENO1 promoter; S.cer ENO1 ter—S. cerevisiae ENO1 terminator;S.cer. URA3 —S. cerevisiae URA3 auxotrophic marker; 2 mu ori—2μ. S.cerevisiae plasmid origin of replication; bla(AmpR)—Amp resistancemarker; pBR322 —E. coli pB322 plasmid origin of replication; TEF1pr—Ashbya gossypii TEF1 promoter; TEF1 ter—A. gossypii TEF1 terminator;ble (Zeo) R—Streptoalloteichus hindustanus ble Zeocin resistance gene.

FIG. 3A depicts CMC and FIG. 3B depicts avicel assay results for EG1candidates expressed in M0509. All EG1 constructs were tested under thecontrol of the ENO1 promoter and terminator. Strain M1322 is expressingan EG from the termite C. formosanus. T. reesei EG1 and T. reesei EG2were included as controls.

FIG. 4 depicts results from a pretreated hardwood (PHW) assay for thetop 6 EG1 candidates, mixed with yeast made, purified, TeCBH1w/TrCBD,and C1CBH2, and Novozyme 188.

FIG. 5 depicts results of a PHW assay for EG1 candidates in the presenceof Novozyme 188.

FIG. 6 depicts results of a SDS-PAGE analysis of the supernatants of (A)the EG2 and (B) the EG3 producing strains. A strain containing a plasmidwith no foreign gene was used as reference strain (REF). The straincontaining the plasmid pRDH180 expressing T.r.eg2, the most successfulEG previously found, was also included.

FIG. 7 depicts results of a CMC and a barley-β-glucan assay. Cultureswere spotted on SC^(−URA) plates containing 0.2% of either CMC (A and B)or barley-β-glucan (C). Numbers indicate the plasmid contained by eachstrain. pRDH180 contained the T. reesii eg2 and served as positivecontrol. Plates were incubated for 3 (A) or 24 (B & C) hours at 30° C.,after which colonies were washed of and the plates were stained with0.1% congo red and de-stained with 1% NaCl.

FIG. 8 depicts results from an assay measuring activity of YPD and SCcultured strains expressing EGs on avicel (24 hours) and CMC (3 hours).A strain containing a plasmid with no foreign gene was used as referencestrain (REF) and the strain expressing T.r.eg2 (pRDH180) was included aspositive control.

FIG. 9 depicts results of a CMC plate assay of EG4, EG5, and EG6 clonesto verify activity expression of the genes.

FIG. 10 depicts PHW assay results for candidate EG4s, EG5s, and EG6s.

FIG. 11 depicts results from experiments with EG4, EG5, EG6, andxyloglucanase candidates by PHW assay. Cultures were grown in 15 mls ofYPD for 2 days at 35 degrees in 50 ml tubes. Cultures were spun down and2 mls of each supernatent was added to 2 mls of PHW components (Negativecontrol is M0544, and M1179 expresses CBH1, CBH2, EG2, and BGL). 4 mg/gof purified enzymes was used as a screening partner in a ratio of40:40:15:5 of CBH1:CBH2:EG2:BGL1.

FIG. 12 depicts results of a SDS-PAGE analysis of the supernatants of(A) xylanase and (B) xylosidase producing strains. A strain containing aplasmid with no foreign gene was used as reference strain (REF). Thestrain containing the plasmid pRDH182 (expressing T.r.xyn2) orcontaining the plasmid pRDH181 (expressing A.n.xlnD) was also included.

FIG. 13 depicts the results of a RBB-xylan assay. Cultures were spottedon SC^(−URA) plates containing 0.2% RBB-xylan. Numbers indicate theplasmid contained by each strain. Plates were incubated for 24 hours at30° C.

FIG. 14A and FIG. 14B depict results of an assay measuring activity ofYPD and SC cultured strains expressing xylanases and xylosidases on 1%birchwood glucuronoxylan (A) and pNPX (B). A strain containing a plasmidwith no foreign gene was used as reference strain (REF).

FIG. 15 depicts results from an assay measuring hydrolytic activity asmeasured by reducing sugar released by mixtures of yeast supernatantsfrom 5% xylan.

FIG. 16 depicts results of a TLC assay measuring sugars released byyeast supernatants from birchwood glucuronoxylan. Std1 containedxylotetrose, xylotriose, xylobiose and xylose; Std2 contained,xylotriose, xylobiose and xylose. 5 μL of reactions 1 to 6 were loaded.

FIG. 17 depicts results of an arabinofuranosidase activity assay withpNPA as substrate.

FIG. 18 depicts results of an esterase activity of candidate enzymes onpNP-acetate.

FIG. 19A and FIG. 19B depict results in a PHW assay on unwashed MS630for various accessory enzymes. Cultures were grown for 3 days at 35degrees in 10 mls YPD with 20 ug/ml zeocin in 50 ml conical tubes. 1 mlof supernatant was added for each candidate, 0.5 ml each of M1457 (BC60xylanase) and M1381 (P.t.r. GH43 xylosidase) plus 2 mls of PHW core mix.Core enzymes added were 1 mg/g of purified CBH1/CBH2/EG2 and 0.2 mg/g ofBGL1.

FIG. 20A, FIG. 20B and FIG. 20C depict results of a PHW assay usingcombinations of accessory enzymes on unwashed MS630 (hardwoodsubstrait). So called “Big 6” enzymes were: 1 mg/g of purified CBH1 andCBH2, 0.4 mg/g purified EG2, and 0.2 mg/g purified BGL, 0.5 mL of eachof M1457 (GH10 xylanase from C. phytofermentens, or BC60—see bacterialenzyme screening below) and M1381 (P.t.r. GH43 xylosidase). These werecombined with PHW and buffer in a total volume of 2 mL and 2 mL ofadditional enzymes were added as tests, split evenly between the enzymes(i.e. 1 mL each of 2 enzymes, or 0.67 mL each of 3 enzymes, etc).Results for glucose and xylose liberated are depicted in panels A and Brespectively.

FIG. 21 depicts results from a xylanase assay of yeast strainsexpressing bacterial (top) and fungal (bottom) enzymes. On the top graphthe numbers mean BC numbers described in Table 7.

FIG. 22A, FIG. 22B, FIG. 22C and FIG. 22D depict results from an assayevaluating the secreted activity on CMC of bacterial endoglucanasesexpressed in yeast. Strains were patched on YPD+Zeo plates (Zeo 250mg/L) for 2 days and inoculated in 600 uL YPD in 96 wp, and grown for 3days at 35° C. at 900 rpm. The standard CMC assay was performed onsupernatants. All strains have M0749 background. The negative control isM0749 transformed with empty expression vector pMU1575. T. reesei EG2 inpMU1575 was used as positive control construct.

FIG. 23 depicts results from a PHW assay with yeast-made bacterialendoglucanases (see Table 7) in the presence of yeast made purified CBH1and CBH2. All wells were supplemented with 3.5 mg/g TS BGL(Novozyme-188) and 2 mg/g TS yeast made purified CBH1+CBH2 (ratio1:1).Supernatant of the strain expressing empty vector was used as negativecontrol.

FIG. 24 depicts results from an assay measuring glucose release from PHWprovided by different combinations of bacterial GH9 EG (T. fusca Cel9A)and fungal GH5 EG (T. reesei EG2). The negative control (empty vector)was added in amount of 2 ml. Compositions of all other samples are shownon the figure. Left side bars depict results from samples that weresupplemented by purified yeast made enzymes (1 mg/g CBH1, 1 mg/g CBH2,0.2 mg/g BGL) plus not purified yeast made xylanase (BC60, 100 ul/well)and xylosidase (M1381, 100 ul/well). Right side bars depict results fromsamples that were supplemented with the same amount of purified CBHsplus 1 mg/g AB BGL.

FIG. 25A and FIG. 25B depict results of an assay of secreted activity onbirchwood xylan for bacterial xylanases expressed in yeast. Strains werepatched on YPD+Zeo plates (Zeo 250 mg/L) for 2 days and inoculated in600 μL YPD in 96 well plate. Plates were then grown for 3 days at 35° C.at 900 rpm. Standard xylose assay (DNS based) was performed on s. Allstrains have M0749 background. The negative control is M0749 transformedwith empty expression vector pMU1575. T. reesei Xyn2 in pMU1575 was usedas positive control construct.

FIG. 26 depicts the results of an assay measuring the effect of yeastmade xylanases on glucose release from PHW by yeast made cellulasesmeasured by PHW assay. Left-side bars depict results from an assay thatwas supplemented with yeast made purified cellulases (CBH1-1 mg/g TS;CBH2-1 mg/g TS; EG2—0.4 mg/g TS, BGL—0.2 mg/g TS) and yeast madeunpurified Pyrenophora tritici-repentis β-xylosidase (GH43, M1381)—50 ulsup/4 ml reaction. M1381 strain expressing xylosidase was grown in YPDin shake flask for 3 days. Right side bars depict results from an assaythat was supplemented with the same amount of yeast made purified CBH1,CBH2 and EG2 plus 1 mg/g TS AB BGL (ME057). The glucose was measured bya glucose hexokinase kit (Sigma). Each experiment was performed intriplicates. Supernatant from a strain expressing empty vector was usedas negative control (NegCon). Supernatant expressing fungal T. reeseiXyn2 was used as positive control.

FIG. 27 depicts results from a xylanase assay in which yeast strainsexpressing T. saccharolyticum xylanase genes were evaluated.

FIG. 28 depicts results from an assay measuring glucose release from PHWprovided by bacterial accessory enzymes in the presence of yeast madeenzymes. A standard PHW assay was performed. Glucose was measured byHPLC. The sample numbers mean BC numbers (see Table 7). All samples wereadded in amount of 2 ml. All samples including NC (negative control)were supplemented with purified 1 mg/gCBH1, 1 mg/gCBH2, 0.4 mg/gEG2, 0.2mg/g BGL; not purified 2.5% (v/v) xylanase (M1457) and 2.5% (v/v).

FIG. 29A and FIG. 29B depict results from an assay measuring glucoserelease from PHW provided by different combinations (pairs) of EGs thatbelong to different GH families Glucose was measured by glucosehexokinase kit. The samples were taken at 27 hrs (A) and 48 hrs (B). Thesample numbers are the GHF numbers (see Tables below). NegCon (NC—emptyvector) supernantant was added in amount of 2 ml. The first bar in eachcolored block is 2 ml of single EG. All other bars in each colored blockrepresent a combination of two different EGs (1 ml each). All samplesincluding NC were supplemented with 1 mg/g CBH1+1 mg/gCBH2+4 mg/g EE.EE—External Enzymes was composed of 3.25 mg/g ME50-2 (cellulaseNovozyme22C, batch# CZP00004, Novozymes); 0.25 mg/g ME54-2 (xylnaseXYN30, batch# EL2007020L, EB Enzymes; 0.25 mg/g ME57 (β-glucosidase ABK,batch# EL2008044L, EB Enzymes; and 0.25 mg/g ME64 (Pectinase F E,batch#1660 05x/lm 401-083-3580, Genencor). MS630 (a pretreated hardwood)was used as substrate. All experiments were performed in triplicate. Themissing bars or the bars without error bars had all or most of therepeats fail.

FIG. 30 depicts results from an assay measuring glucose release from PHWprovided by different combinations (triplets) of EGs that belong todifferent GH families Glucose was measured by GHK kit. The samples weretaken at 48 hrs. The sample numbers are GHF numbers (see Tables below).The negative control (NC—empty vector) and other single EGs supernatantswere added in amount of 2 ml. In samples with two EGs, 1 ml of eachsupernatant was added. In samples with three EGs 0.666 ml of eachsupernatant was added. All samples including NC were supplemented with 1mg/g CBH1+1 mg/gCBH2+4 mg/g EE. MS630 was used as substrate (apretreated hardwood). All experiments were performed in triplicate. Thebar without error bars had two repeats fail.

FIG. 31A, FIG. 31B, FIG. 31C and FIG. 31D depict results from an assaymeasuring glucose release from PHW provided by different combinations ofEGs that belong to different GH families. Glucose was measured by aglucohexokinase kit. The samples were taken at 24 (A), 48 (B), 72 (C)and 96 (D) hrs. The sample numbers are GHF numbers (see Tables). Thenegative control (NC—empty vector) and other single EGs supernatantswere added in amount of 2 ml. In samples with two EGs 1 ml of eachsupernantant was added. In samples with three EGs 0.666 ml of eachsupernatant was added. In samples with four EGs 0.5 ml of eachsupernatant was added. All samples including NC were supplemented with 1mg/g CBH1+1 mg/gCBH2+EE (EE composition, see above). EE was added at 2mg/g TS (blue bars) or 4 mg/g TS (purple bars). All experiments wereperformed in triplicate.

FIG. 32 depicts a time course of glucose release from PHW provided byselected samples from FIG. 31.

FIG. 33 depicts a CEN vector with a Gal promoter upstream of thecentromere and an ARS replication origin (another 2μ origin is alsopresent to fire replication at multiple points for large vectors). Thefour endoglucanases have unique promoters driving them. Thepromoter/EG/terminator cassettes were PCR amplified from existingvectors and incorporated into NotI digested pMU1943. The right handpanel shows the activity of 6 separate colonies picked from the YMLtransformation plate, which all demonstrated EG activity.

FIG. 34A and FIG. 34B depict CEN vectors built for testing the abilityto assemble large constructs. M1634 contains the CEN with 7 genes (23kB), and M1635 contains the CEN with 11 genes (M1635).

FIG. 35 depicts results from an assay measuring CMC activity forcolonies picked from selective and non-selective plates after growth ofthe starting culture in YPD or YP-Galactose. Activity is comparablebefore and after galactose treatment in colonies from high antibioticresistance plates. Colonies treated with galactose and plated on YPDwithout hygromycin show a large variation as seen from the error barsindicating that the CEN vector is functioning as expected duringgalactose growth.

FIG. 36 depicts results from a CMC assay on strains expressing CEN6vector passaged twice (about 10 generations) in YPD without antibiotic.The CMC activity is comparable after passaging for about 10 generationsin YPD without antibiotic. It should be noted that FIG. 35 shows the CMCassay data after only an hour, whereas the CMC assay before passagingthe strains is for a 1.5 hour time point.

FIG. 37 depicts an assay which is a comparison between thetop-performing colonies from YPD/zeocin (100) and YPD/zeocin (50) platesat various dilutions.

FIG. 38 depicts results from a PHW assay with yeast produced enzymesalone. M1179 (Strain with core cellulases CBH1/CBH2/EG2/BGL1) was usedalong with CEN strain expressing 4 EGs (EG1, 4, 5 and 6) strain M1377(EG3) and M1050 (cel9A).

FIG. 39 depicts conversion of xylan to ethanol by several strains of S.cerevisiae expressing xylanase alone, xylosidase alone, or a combinationof the two enzymes.

FIG. 40 depicts a genetic construct used to co-express xylanase andxylosidase via integration at the rDNA loci.

FIG. 41 depicts a map of the episomal 2-μ yeast expression vector usedfor expression of genes from Tables 15-17. S.cer ENO1 pr—S. cerevisiaeENO1 promoter; S.cer Invertase SP—S. cerevisiae Invertase signalpeptide; S.ser ENO1 ter—S. cerevisiae ENO1 terminator; S.cer. URA3 —S.cerevisiae URA3 auxotrophic marker; 2 mu ori—2μ S. cerevisiae plasmidorigin of replication; bla(AmpR)—Amp resistance marker; pBR322 —E. colipB322 plasmid origin of replication; TEF1 pr—Ashbya gossypii TEF1promoter; TEF1 ter—A. gossypii TEF1 terminator; ble (Zeo)R—Streptoalloteichus hindustanus ble Zeocin resistance gene.

FIG. 42 depicts secreted activity of strains expressing new syntheticgenes measured by Starch-DNS (top), Starch-GHK (middle), and Maltose(bottom) assays. All genes are described in Tables 15 and 16. All geneswere inserted between PacI/AscI of pMU1575 2μ expression vector andtransformed into M1744 strain. Transformants were grown in YPD for 3days and supernatants were analyzed for activity. “CO”—codon optimizedfor yeast synthetic genes; others—PCRed from genomic DNA or cDNA.

FIG. 43 depicts starch activity of yeast made amylolytic enzymes incombination with yeast made AE8. Supernatants of strains grown for 3days in YPD were mixed with supernatant of AE8 expressing strain at50:50 ratio. In the first sample AE8 supernatant was 100%. Supernatantof M0509 was used as negative control. “CO”-codon optimized for yeastsynthetic genes; others—PCRed from genomic DNA or cDNA.

FIG. 44 depicts a corn mash assay for new secreted genes individuallyand in combination with AE8. Supernatants of strains grown for 3 days inYPD were mixed with supe of AE8 expressing strain at 50:50 ratio.Supernatant of M0509 was used as negative control.

FIG. 45 depicts the effect of arabinases (top) and xylanases (bottom)added to AE8 on glucose release from non pretreated corn fiber.Supernatants of strains grown for 3 days in YPD were mixed withsupernatant of AE8 expressing strain at 50:50 ratio. Supernatant ofM0509 was used as negative control. Arabinases are described in Table 16(AE67-78). ‘BC″’ genes are described in Table 7. “BCTsX1” is theputative xylanase gene PCR amplified from Thermoanaerobacteriumsaccharolyticum genomic DNA based on genome sequence obtained atMascoma.

FIG. 46 depicts the expression of amylolytic enzymes in differentindustrial strains. The expression level of amylases AE3, AE8, and AE49(see Table 16) was evaluated by activity of supernatants on maltose. Allgenes were subcloned into pMU1575 2μ expression vector by yeast mediatedligation and transformed into one of three strains. Transformants weregrown in YPD for 3 days and supernatants were analyzed for activity byMaltose assay. Four transformants were analyzed for each transformation.

FIG. 47 depicts expression constructs used for random integration strainconstruction (top). P—S. cerevisiae promoter; t—S. cerevisiaeterminator; URA3 —S. cerevisiae URA3 marker; D—delta integration sites;“CO”—codon optimized synthetic genes. Combinations of genes used forrandom integration (bottom). Genes used in each combination are markedgray.

FIG. 48 depicts the secreted activity on starch of strains built byrandom integration. Supernatants of strains grown for 3 days in YPD wereused in starch-DNS assay. Ura—transformants were selected from SD-URAplates; Starch—transformants were selected from YM-Starch plates (1×YNBplus 0.5% starch); Controls—strains do not express amylases. CBPstrain-M1973 was used as a positive control. The same experiment wasrepeated twice in duplicates: 1^(st) experiment—top; 2^(nd)experiment—bottom.

FIG. 49 depicts a scheme of directed integration strain constructionapproach with negative selection marker FCY1 used as integration site.Amylolytic strains M1973 and M2016 expressing glucoamylases AE8 and/orAE9 were used as examples. The expression cassettes flanking regions ofFCY were integrated into FCY1 locus (position ˜677162 on chromosome 16)of industrial strain M0139 as PCRed DNA fragments with overlapping ends.The host M0139 is a diploid, therefore each expression cassette wasintegrated in two copies. The 2-μ plasmid with Hyg marker wasco-transformed with PCR products. The transformants were firstcultivated in liquid YPD+Hyg media overnight and then plated on mediawith FCY knock-out selective compound 5-fluorocytosine. Precultivationon media with antibiotic increases efficiency of double FCY1 knock-out.

FIG. 50 depicts integration of additional copies of glucoamylase into agenomic site such as an Adenine-phosphoribosyltransferase 2 (APT2)locus.

FIG. 51 depicts a scheme of directed integration strain constructionapproach with universal integration site. Amylolytic strain M2022expressing multiple copies of glucoamylases AE8 and AE9 was used as anexample. In the first round of transformation (top) four additionalglucoamylase expression cassettes together with APT2 flanking regions,dominant markers (Nat and Kan) and FCY1 marker were integrated into APT2locus (position ˜1345055 chromosome 14) into industrial strain M1973(already expressing 4 glucoamylase copies, see FIG. 50) as PCRed DNAfragments with overlapping ends. The transformants were plated onYPD+Nat+Kan plates that allow growth only for cells that have bothdominant markers integrated into different copies of chromosome. In thesecond round of transformation (middle) the transformants selected forthe high amylolytic activity by Starch-DNS assay were transformed withtwo PCR products that have overlapping ends: 5′-APT2 flanking region and5′ part of AE9 expression cassette. The transformants were patched on5-fluorocytosine containing media that allows selection for lack ofFCY1. On the bottom of the figure the final APT2 integration locus ofM2022 shown. It also shows which S. cerevisiae promoters (pr) andterminators (ter) were controlling expression of newly added AE8 andAE9.

FIG. 52 depicts ethanol produced by amylolytic yeast without exogenousglucoamylase from liquefied corn mash. The numbers are average oftriplicate runs and error bars are 1 std. Inoculum of 0.1 g/L was used.Fermentations were performed in 250 mL sealed shake flasks with a totalfermentation mass of 50 g on corn mash obtained from Valero bio-refineryat 30% solids (TS) at a fermentation temperature of 32° C. at a shakingspeed of 125 rpm. The fermentations were performed using 500 ppm urea asthe only nutrient source. Standard dose (0.45 AGU/g TS) of commercialglucoamylase (Spirizyme Ultra, Novozymes) was added to the controlstrain M0139. All other strains were fermented without any exogenousenzymes added. The ethanol produced after 60 h is shown.

FIG. 53 depicts ethanol produced by amylolytic yeast without exogenousglucoamylase from non-liquefied corn mash. 50 g flask runs on raw starch(corn ground w/2 mm screen Wiley Mill); raw corn slurry 30% solids;0.006 mg/ml Pen G; 0.1 gDCW/l inoculum; T=35° C. for 24 hrs followed by32° C. Average of duplicate flasks shown. The fermentations wereperformed using 500 ppm urea as the only nutrient source. Standard dose(0.45 AGU/g TS) of commercial glucoamylase (Speezyme, Genencor Inc.) wasadded to the control strain M0139. All other strains were fermentedwithout any exogenous enzymes added.

FIG. 54 depicts the adaptation of amylolytic M1973 strain by serialtransfer. 1973—Original M1973 strain from freezer stock; 1973A—AdoptedM1973 strain. The strains were evaluated by fermentation on 30% or 35%TS corn mash (first number) at 32° C. or 35° C. (second number). Datashown for 48 h time point.

FIG. 55 depicts an example of a process flow sheet with CBP yeaststrains. Ground corn mash is used as a substrate. Two yeast CBP strainsare used in the process and cultured separately, S1 and S2. Liquefiedcorn pre-treated with alpha-amylases is fermented by yeast strain S1. S1has an optimal set of amylases and accessory enzymes engineered toefficiently convert starch into glucose without any exogenous enzymesadded. After distillation the stillage is being pre-treated andfermented by strain S2. S2 has a cellulolytic set of enzymes engineeredand optimized for corn fiber conversion as well as xylose and arabinosepathways.

FIG. 56 depicts PCR genotyping of industrial yeast strains genomic DNA(Ness et al. 1993). 1 kb—NEB 1 kb ladder. A—M0139 like pattern; B—M2390like pattern.

FIG. 57 depicts growth of industrial yeast strains at 41° C. Strainswere streaked for singles on YPD plate and incubated at 41° C. for 4days.

FIG. 58 (Top) depicts maximum growth rate at 41° C. in YPD of industrialstrains described in Table 20. Growth rate measured by plate readerSynergy 2 (BioTek) following manufacture's instructions. Bottom—Cornflour fermentation in shake flasks at 72 h of industrial strainsdescribed in Table 20. Raw corn flour was used as substrate.Fermentation was performed at 35% of total solids; at the temperature of35° C. for 24 h followed by 32° C. for the rest of fermentation. Strainsmarked with “*” were done in separate experiment at similar conditionsbut at 33% of total solids. Full commercial dose of exogenous GA wasadded to all strains at concentration 0.6 AGU/g of total solids.Experiment was done in duplicates. Commercial enzyme Spirizyme Ultra(Novozymes) was used as exogenous glucoamylase. Ethanol was measured byHPLC.

FIG. 59 depicts a map of expression construct used to transformdifferent industrial hosts. ENO1—S. cerevisiae ENO1 promoter; AE9CO—codon optimized for S. cerevisiae Saccharomycopsis fibuligeraglucoamylase gene (NCBI#CAC83969.1); S.cer ENO1 ter—S. cerevisiae ENO1terminator; PDC1—S. cerevisiae PDC1 terminator; ADH1—S. cerevisiae ADH1promoter; TEF—S. cerevisiae TEF2 promoter; nat1-Streptomyces nourseinat1 genes that confers resistance to antibiotic Nourseothricin; TRI1—S.cerevisiae TRI1 terminator. DNA fragments were PCRed separately andrecombined in vivo during yeast transformation.

FIG. 60 depicts secreted amylolytic activity of industrial strains(Table 20) transformed with 4 copies of Saccharomycopsis fibuligeraglucoamylase gene (NCBI#CAC83969.1). Top panel shows the names of hoststrains. Activity was measured by Starch assay. Several transformantswere picked for each host. Supernatant of untransformed M0139 strain wasused as negative control (C).

FIG. 61 depicts corn flour fermentation in shake flasks at 72 h ofindustrial strains and their transformants engineered to express 4copies of Saccharomycopsis fibuligera glucoamylase gene(NCBI#CAC83969.1). Raw corn flour was used as a substrate. The strainsare described in the tables 20 and 22. Fermentation was performed at 35%of total solids; at the temperature of 35 C for 24 h followed by 32° C.for the rest of fermentation. Exogenous GA was added to all strains atconcentration 0.3 AGU/g of solids. Transformed strains were done induplicates. Host strains were done in singles. Commercial enzymeSpirizyme Ultra (Novozymes) was used as exogenous glucoamylase. Ethanolwas measured by HPLC.

FIG. 62 depicts corn mash fermentation in shake flasks at 48 h ofindustrial strains and their transformants engineered to express 4copies of Saccharomycopsis fibuligera glucoamylase gene(NCBI#CAC83969.1). Liquefied corn pre-treated with alpha-amylases fromconventional plant was used as substrate. The strains are described inthe tables 20 and 22. Fermentation was performed at 35% of total solidsand 35° C. Exogenous GA was added to all strains at concentration 0.3AGU/g of solids. The experiment was done in duplicates. Commercialenzyme Spirizyme Ultra (Novozymes) was used as exogenous glucoamylase.Ethanol was measured by HPLC.

FIG. 63 depicts secreted amylolytic activity of M2390 transformantsengineered to express 4 copies of AE9—Saccharomycopsis fibuligeraglucoamylase gene (NCBI#CAC83969.1). About 1000 transformants werescreened by Starch assay. This experiment shows repeated Starch assaydata for 30 the most active transformants. Experiment was done intriplicates. Supernatant of untransformed M2390 strain was used asnegative control. Strains M2111 and M2395 were used as positive control(see Tables 20 and 21 for strains description).

FIG. 64 depicts corn mash fermentation in minivials at 72 h of M2390transformants engineered to express 4 copies of AE9—Saccharomycopsisfibuligera glucoamylase gene (NCBI#CAC83969.1). Seventeen besttransformants from amylolytic activity screen (FIG. 63) were selectedfor this experiment. Fermentation was performed at 30% of total solidsand 30° C. Exogenous GA was added to the untransfomed M2390 strain only,at concentration 0.3 AGU/g of solids. The experiment was done induplicates. M2111, M2395 and M2390 strains were used as controls (seetables 20 and 21 for strains description). Commercial enzyme SpirizymeUltra (Novozymes) was used as exogenous glucoamylase. Ethanol wasmeasured by HPLC.

FIG. 65 depicts corn flour fermentation in minivials at 72 h of M2390transformants engineered to express 4 copies of AE9—Saccharomycopsisfibuligera glucoamylase gene (NCBI#CAC83969.1). Seventeen besttransformants from amylolytic activity screen (FIG. 63) were selectedfor this experiment. Fermentation was performed at 30% of total solidsand 30° C. Exogenous GA was added to the untransfomed M2390 strain atconcentration 0.3 AGU/g of solids and at 0.1 AGU/g to all other strains.The experiment was done in duplicates. M2111, M2395 and M2390 strainswere used as controls (see Tables 20 and 21 for strains description).Commercial enzyme Spirizyme Ultra (Novozymes) was used as exogenousglucoamylase. Ethanol was measured by HPLC.

FIG. 66 depicts corn flour fermentation in shake flasks at 72 h of M2390transformants engineered to express 4 copies of AE9—Saccharomycopsisfibuligera glucoamylase gene (NCBI#CAC83969.1). Seven best transformantsfrom minivials fermentation screen (FIGS. 64-65) were selected for thisexperiment. Fermentation was performed at 33% of total solids at thetemperature of 35° C. for 24 h followed by 32° C. for the rest offermentation. Exogenous GA was added to the untransfomed M2390 strain atconcentration 0.6 AGU/g of solids and at 0.1 AGU/g to all other strains.The experiment was done in duplicates. Commercial enzyme Spirizyme Ultra(Novozymes) was used as exogenous glucoamylase. Ethanol was measured byHPLC.

FIG. 67 depicts time course of liquefied conventional corn mashfermentation in shake flasks of M2691 strain—the best M2390 transformantengineered to express 4 copies of AE9—Saccharomycopsis fibuligeraglucoamylase gene (NCBI#CAC83969.1). Transformant P10-19 (FIG. 66) wasre-named as M2691. Fermentation was performed at 32.5% of total solidsat the temperature of 35° C. for 24 h followed by 32° C. for the rest offermentation. Exogenous GA was added to the untransfomed M2390 strainonly, at concentration 0.3 AGU/g of solids. The experiment was done induplicates. Commercial enzyme Spirizyme Ultra (Novozymes) was used asexogenous glucoamylase. Ethanol was measured by HPLC.

FIG. 68 depicts time course of raw corn flour fermentation in shakeflasks of M2691 strain—the best M2390 transformant engineered to express4 copies of AE9-Saccharomycopsis fibuligera glucoamylase gene(NCBI#CAC83969.1). Transformant P10-19 (FIG. 66) was re-named as M2691.Fermentation was performed at 33% of total solids at the temperature of35° C. for 24 h followed by 32° C. for the rest of fermentation.Exogenous GA was added to the untransfomed M2390 strain at concentration0.6 AGU/g of solids and at 0.1 AGU/g to M2691. The experiment was donein duplicates. Commercial enzyme Spirizyme Ultra (Novozymes) was used asexogenous glucoamylase. Ethanol was measured by HPLC.

FIG. 69 depicts exogenous glucoamylase dose response for untransformedM2390 strain, low GA producer M2395 strain, and high GA producer M2519(P6-65). Corn flour shake flasks fermentation was performed at 35% oftotal solids at the temperature of 35° C. for 24 h followed by 32° C.for the rest of fermentation. The experiment was done in duplicates.Commercial enzyme Spirizyme Ultra (Novozymes) was used as exogenousglucoamylase. Ethanol and glucose were measured by HPLC.

FIG. 70 depicts stability test of two M2390+AE9 transformants, M2519(top) and M2691 (bottom). Both strains were propagated in YPD. Strainswere grown to stationary phase and passaged with 100× dilution 11 times(1 passage—about 9 generations). Several samples between passages werestocked. All samples and original strain were plated and inoculatedtogether and activity on starch was measured in the same assay.Experiment was done in triplicates.

FIG. 71A depicts Pullulan, FIG. 71B depicts Xylan and FIG. 71C depictsPectin assays of yeast secreted enzymes (Table 23). The genes wereexpressed under ENO1 promoter and terminator from 2-micron plasmidpMU1575. The genes were inserted between PacI/AscI sites of pMU1575either by cloning or yeast mediated ligation. Expression contracts weretransformed into an industrial background Mascoma strain M1744 andselected on minimal URA deficient media. Four colonies were analyzed foreach transformation. Transformants were grown in YPD for 3 days andsupernatants were analyzed for activity. Supernatant of non-transformedstrain M0139 (M1744 derived from M0139 through URA3 gene deletion) wasused as negative control. In Pectin assay C—commercial pectinaseMultifect (Genencor) diluted 10× by citrate buffer was used as positivecontrol (5 μl used in assay).

FIG. 72 depicts corn syrup assay of yeast made enzymes. CBH1, CBH2, EG2,BGL, XYL, and XLD were HPLC purified proteins. For other enzymes yeaststrains expressing enzymes were grown for 3 days in YPD and supernatantswere used as enzyme source (Table 24). B4—CBH1+CBH2+EG2+BGL;B6—CBH1+CBH2+EG2+BGL+XYL+XLD. Amounts of purified enzymes used in assayare summarized in the Table 25. 250 μl of M0139 (top) or M2111 (bottom)supe was added to all samples. Other supernatant derived enzymes wereadded in amount of 250 μl. In no other supernatant enzymes needed in thesample, M0139 supernatant was added instead. For AE10+AE35 sample 125 μlof each supernatant was added in addition to 250 μl of M0139 or M2111supernatant. NC-no other enzymes added except for M0139 or M2111supernatant.

FIG. 73 depicts a map of the episomal 2-micron yeast expression vectorpMU2382 used for construction of delta integration expression cassetteswith genes in Table 26. Gene of interest under control of S. cerevisiaestrong constitutive promoter and terminator was inserted between URA3and Delta2 fragments of pMU2382 vector digested with BamHI and EcoRI.The cassette was inserted by yeast mediated ligation in the sameorientation as URA3. S.ser. URA3 —S. cerevisiae URA3 auxotrophic marker;2 mu ori—2 micron S. cerevisiae plasmid origin of replication;bla(AmpR)—Amp resistance marker; pBR322 —E. coli pB322 plasmid origin ofreplication, delta 1 and delta 2—fragments of S. cerevisiae delta sites.

FIG. 74 depicts an example of corn flour assay of M2125 transformed withsome genes and gene combos from Table 26. Transformations (T) aredescribed in the Table 27. Number after dash means colony number forthis transformation. Transformants that are highlighted were selectedfor screening by fermentation. BC60—M1744 strain expressing only BC60 on2μ plasmid under ENO1 promoter. M2125—parental strain (M2111 with URA3knockout). Untransformed M0139 strain was used as negative control.

FIG. 75 depicts shake flask fermentation on homemade corn mash ofstrains expressing additional to AE9 saccharolytic enzymes. Strainsselected based on highest ethanol titers reached in minivial corn mashfermentation assay. Homemade mash was used. The strains are described inthe Table 28. Fermentation was performed at 30% of total solids and 32°C. Exogenous enzyme was added to the untransfomed M0139 strain only, atconcentration 0.3 AGU/g of solids. Parental M2111 strain was used asbackground control. The experiment was done in duplicates. Commercialenzyme Spirizyme Ultra (Novozymes) was used as exogenous glucoamylase.Ethanol was measured by HPLC.

FIG. 76 depicts shake flask fermentation on corn flour of strainsexpressing additional to AE9 saccharolytic enzymes. Strains selectedbased on highest ethanol titers reached in minivial corn flourfermentation assay. The strains are described in the Table 29.Fermentation was performed at 30% of total solids and 32° C. Exogenousenzyme was added to the untransfomed M0139 strain at concentration 0.3AGU/g of solids and at 0.1 AGU/g to all other strains. Parental M2111strain was used as background control. The experiment was done induplicates. Commercial enzyme Spirizyme Ultra (Novozymes) was used asexogenous glucoamylase. Ethanol and sugars were measured by HPLC.Potential ethanol was calculated based on glucose concentration (addedtheoretical ethanol from unconsumed glucose).

FIG. 77 depicts shake flask fermentation on homemade corn mash (top) andcorn flour (bottom) of strains expressing AE9 only. The strains wereresult of repeating the same transformation as was done in M2111construction with consequent screening of 1000 colonies for activity onstarch. Strains for this shake flask experiment were selected based onhighest ethanol titers reached in minivial corn homemade mash and flourfermentation assays. The strains are described in Tables 30 and 31.Fermentation was performed at 30% of total solids and 32° C. Exogenousenzyme was added to the untransfomed M0139 strain at concentration 0.3AGU/g of solids. In corn flour experiment exogenous enzyme was alsoadded to all other strains at concentration 0.1 AGU/g of solids.Previously constructed M2111 strain was included for comparison. Theexperiment was done in duplicates. Commercial enzyme Spirizyme Ultra(Novozymes) was used as exogenous glucoamylase. Line—protein (AE9)secreted by the strains after 3 days growth in YPD shake flasks(separate from fermentation experiment). Ethanol and proteinconcentration were measured by HPLC.

FIG. 78 depicts shake flask fermentation on industrial corn mash of thebest strains from shake flask screening experiments on homemade mash andcorn flour (FIGS. 75-77). The strains are described in Table 32.Fermentation was performed at 30% of total solids and 32° C. Exogenousenzyme was added to the untransfomed M0139 strain only, at concentration0.3 AGU/g of solids. M2111 strain was included for comparison. Theexperiment was done in duplicates. Commercial enzyme Spirizyme Ultra(Novozymes) was used as exogenous glucoamylase. Ethanol and sugarsconcentration were measured by HPLC. Potential ethanol was calculatedbased on glucose concentration (added theoretical ethanol fromunconsumed glucose).

FIG. 79 depicts shake flask fermentation on industrial corn mash of thebest strains from shake flask screening experiments on homemade mash andcorn flour (FIGS. 75-77). The strains are described in Table 33.Fermentation was performed at 30% of total solids and 32° C. Exogenousenzyme was added to the untransfomed M0139 strain only, at concentration0.3 AGU/g of solids. M2111 strain was included for comparison. Theexperiment was done in duplicates. Commercial enzyme Spirizyme Ultra(Novozymes) was used as exogenous glucoamylase. Ethanol and sugarsconcentration were measured by HPLC. Potential ethanol was calculatedbased on glucose concentration (added theoretical ethanol fromunconsumed glucose).

FIG. 80 depicts stability test of M2111 strain built by directedintegration (top) and strains built by random integration (bottom). Thestrains were propagated in YPD, grown to stationary phase and passagedwith 100× dilution 11 times (1 passage—about 9 generations). Severalsamples between passages were stocked. All samples and original strainwere plated and inoculated together and activity on starch was measuredin the same assay. Random strains are described in Table 32. Theexperiment was done in triplicates.

FIG. 81 depicts different possible strategies for directed strainsconstruction. Top—one site integration strategy; bottom—multiple sitesintegration strategy. In one site strategy negative markers alternate ineach transformation round and all expression cassettes are integratedinto the same locus next to each other. In multiple sites strategypositive and negative markers alternate with each other and in eachround of transformation the expression cassette can be integrated intoany site on chromosome.

FIG. 82 depicts a schematic of TeCBH1+HgCBD expression construct forintegration at the δ sites in S. cerevisiae.

FIG. 83 depicts assay of supernatants containing cellulases onpretreated hardwood made by several strains of S. cerevisiae.Supernatants were incubated with pretreated hardwood at 4% total solids,an exogenous cellulase preparation at a 2 mg enzyme/g total solidsloading in the PHW assay. Accumulation of glucose in the reaction wasmeasured by HPLC.

FIG. 84 depicts a comparison of cellulolytic strains containing eitherjust one enzyme (CBH2, M1873), or seven enzymes (M2232) to the controlnon-cellulase producing M1577 for ethanol production in SSF. Bothunwashed pretreated hardwood, and alkaline washed pretreated hardwoodsubstrates were used. Data is presented from 160 hours of fermentation.

FIG. 85 depicts SDS-PAGE (left) and Western blot (right) of yeast madealpha-glucuronidase. Alpha-glucuronidase, GH67 was PCR amplified fromPichia stipitis genomic DNA and cloned +/−C-terminal Histidine tag.Colonies from transformations were grown in yeast extract (10 g/L),peptone (20 g/L), and glucose (20 g/L)+200 μg/mL Zeocin, pH 7.0 in 50 mLvented conical tubes for 48-60 hours. Cultures supernatants werefiltered through a 2 μm PE filter and concentrated approximately 20-foldin a 10,000 Da molecular weight cut off filter. Protein quality wasscreened via SDS-PAGE electrophoresis under non-reducing conditions andstained with Coomassie Blue dye (left) or examined by Western Blot(right) using an anti-Histidine primary antibody and alkalinephosphatase conjugated secondary antibody (only His tagged constructsvisualized).

FIG. 86 depicts xyloglucanase activity on AZCL-xyloglucan agar plates.Equal amounts of culture were spotted onto SC agar plates containing0.5% AZCL (Azurine-Crosslinked) tamarind xyloglucan Megazyme catalog #I-AZXYG. Xyloglucanase activity is indicated as blue zones such as thosestrains transformed with pMU2856 and pMU2858+/−His tag. REF refers tocontrol MO1744 background strain supernatant.

FIG. 87 depicts xyloglucanase activity in AZCL-xyloglucan. 70 μL ofsupernatant of 3 day old 2×SC^(−ura) cultures were added to 280 μL of 50mM Na-Acetate buffer (pH 5.0) containing 0.5% AZCL (Azurine-Crosslinked)tamarind xyloglucan Megazyme catalog # I-AZXYG in a deep-well microtiterplate. The plate was incubated in a microtiter plate shaker at 35° C. at800 rpm agitation. Samples of 100 μL were taken at 0, 60 and 180 minutesof incubation, spun down at 3000 rpm (2 minutes) after which 50 μL ofthe supernatant was placed in a fresh microtiter plate and the OD at 600nm was determined so that the increased OD over time could be measured.REF refers to control MO1744 background strain.

FIG. 88 depicts SDS-PAGE (left) and Western (right) analysis of yeastexpressed xyloglucanases +/−His tags. Three days old cultures in doublestrength SC-URA media buffered to pH6.0 (3 mL cultures in test tubesincubated at 30° C. on rotary wheel) were centrifuged and supernatantsassayed by loading 15 μL (+5 μL loading buffer) onto 10% SDS-PAGE gels.REF refers to control MO1744 background strain supernatant.

FIG. 89 depicts SDS-PAGE analysis of esterases expressed inSaccharomyces cerevisiae. Three day old cultures in double strengthSC^(−URA) media buffered to pH6.0 (3 mL cultures in test tubes incubatedat 30° C. on rotary wheel) were centrifuged and supernatants assayed byloading 15 μL (+5 μL loading buffer) onto 10% SDS-PAGE gels and silverstained. REF refers to control MO1744 background strain supernatant.

FIG. 90 depicts 1-Napthyl-acetate esterase assay of yeast madeesterases. Experiment was performed in duplicates. REF refers to controlM1744 background strain supernatant.

FIG. 91 depicts Alpha-galactosidase activity assay with yeast madealpha-galactosidases. Experiment was performed in duplicates. REF refersto control M1744 background strain supernatant.

FIG. 92 depicts Western blot analysis of T. reesei alpha-galactosidase(AGL3) +/−His tag expression in Saccharomyces cerevisiae. Colonies fromtransformations were grown in yeast extract (10 g/L), peptone (20 g/L),and glucose (20 g/L)+200 ug/mL Zeocin, pH 7.0 in 50 mL vented conicaltubes for 48-60 hours. Cultures supernatants were filtered through a 2μm PE filter and concentrated approximately 20-fold in a 10,000molecular weight cut off filter. Protein quality was screened viaSDS-PAGE electrophoresis under non-reducing conditions and examined byWestern Blot using an anti-Histidine primary antibody and alkalinephosphatase conjugated secondary antibody (only His tagged constructsvisualized).

FIG. 93 depicts SDS-PAGE analysis of alpha-galactosidases expression inSaccharomyces cerevisiae. Three day old cultures in double strengthSC^(−URA) media buffered to pH6.0 (3 mL cultures in test tubes incubatedat 30° C. on rotary wheel) were centrifuged and supernatants assayed,and 15 μL (+5 μL loading buffer) was loaded onto 10% SDS-PAGE gels andsilver stained.

FIG. 94 depicts a 2% total solids PWH assay with different combinationsof commercial and yeast made purified enzymes and the resultant glucoserelease. The assay plate was incubated at 38° C. and samples wereremoved at various time points for HPLC analysis on the BioRad 87Hcolumn

FIG. 95 depicts a 2% total solids PWH assay with different combinationsof commercial and yeast made purified enzymes and the resultant glucoserelease. The assay plate was incubated at 38° C. and samples wereremoved at various time points for HPLC analysis on the BioRad 87Hcolumn.

FIG. 96 depicts a 2% total solids PWH assay with different combinationsof commercial and yeast made purified enzymes and the resultant glucoserelease. The assay plate was incubated at 38° C. and samples wereremoved at various time points for HPLC analysis on the BioRad 87Hcolumn.

FIG. 97 depicts a 2% total solids paper sludge assay of differentcombinations of yeast made purified enzymes and the resultant glucoserelease. The assay plate was incubated at 38° C. and samples wereremoved at various time points for HPLC analysis on the BioRad 87Hcolumn.

FIG. 98 depicts a 2% total solids paper sludge assay of differentcombinations of yeast made purified enzymes and the resultant xyloserelease. The assay plate was incubated at 38° C. and samples wereremoved at various time points for HPLC analysis on the BioRad 87Hcolumn.

FIG. 99 depicts final ethanol titers (92 hours) for 2 differentindustrial paper sludges SSF. Sludge 1—first 5 bars; Sludge 2—last 5bars. Washed (1M Citric acid) 2% solids paper sludges were used. StrainM2108 was inoculated at 1.1 g/l. Fermentation was performed at pH5.0,35° C., 220 rpm, 92 hrs.

FIG. 100 depicts ethanol and potential ethanol titers achieved on 30% TScorn flour with 0.1 AGU/g TS exogenous gluco-amylase. The control strain(M0139) has a full dose (0.3 AGU/g TS) of gluco-amylase.

FIG. 101 depicts ethanol and potential ethanol titers at 72 hours forxylanase and accessory enzyme screen on 30% TS corn flour (ELN afoster2corn-090).

FIG. 102 depicts glucose, xylose and arabinose released from ahydrolysis of 2% TS pretreated wet cake.

FIG. 103 depicts hydrolysis yields from 190° C., 10 minutes waterpretreated coarse fiber and 1% sulfuric acid pretreated coarse fiber.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed methods and materials are useful generally in the field ofengineered yeast.

Definitions

A “vector,” e.g., a “plasmid” or “YAC” (yeast artificial chromosome)refers to an extrachromosomal element often carrying one or more genesthat are not part of the central metabolism of the cell, and is usuallyin the form of a circular double-stranded DNA molecule. Such elementsmay be autonomously replicating sequences, genome integrating sequences,phage or nucleotide sequences, linear, circular, or supercoiled, of asingle- or double-stranded DNA or RNA, derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction which is capable of introducing a promoter fragmentand DNA sequence for a selected gene product along with appropriate 3′untranslated sequence into a cell. Preferably, the plasmids or vectorsof the present invention are stable and self-replicating.

An “expression vector” is a vector that is capable of directing theexpression of genes to which it is operably associated.

The term “intergrated” as used herein refers to genetic elements thatare placed, through molecular biology techniques, into the genome of ahost cell. For example, genetic elements can be placed into thechromosomes of the host cell as opposed to in a vector such as a plasmidcarried by the host cell. Methods for integrating genetic elements intothe genome of a host cell are well known in the art and includehomologous recombination.

The term “heterologous” when used in reference to a polynucleotide, agene, a polypeptide, or an enzyme refers to a polynucleotide, gene,polypeptide, or an enzyme not normally found in the host organism.“Heterologous” also includes a native coding region, or portion thereof,that is removed from the source organism and subsequently reintroducedinto the source organism in a form that is different from thecorresponding native gene, e.g., not in its natural location in theorganism's genome. The heterologous polynucleotide or gene may beintroduced into the host organism by, e.g., gene transfer. Aheterologous gene may include a native coding region that is a portionof a chimeric gene including non-native regulatory regions that isreintroduced into the native host. Foreign genes can comprise nativegenes inserted into a non-native organism, or chimeric genes. Aheterologous polynucleotide, gene, polypeptide, or an enzyme may bederived from any source, e.g., eukaryotes, prokaryotes, viruses, orsynthetic polynucleotide fragments. The term “heterologous” as usedherein also refers to an element of a vector, plasmid or host cell thatis derived from a source other than the endogenous source. Thus, forexample, a heterologous sequence could be a sequence that is derivedfrom a different gene or plasmid from the same host, from a differentstrain of host cell, or from an organism of a different taxonomic group(e.g., different kingdom, phylum, class, order, family genus, orspecies, or any subgroup within one of these classifications). The term“heterologous” is also used synonymously herein with the term“exogenous.”

The term “domain” as used herein refers to a part of a molecule orstructure that shares common physical or chemical features, for examplehydrophobic, polar, globular, helical domains or properties, e.g., a DNAbinding domain or an ATP binding domain. Domains can be identified bytheir homology to conserved structural or functional motifs. Examples ofcellobiohydrolase (CBH) domains include the catalytic domain (CD) andthe cellulose binding domain (CBD).

A “nucleic acid,” “polynucleotide,” or “nucleic acid molecule” is apolymeric compound comprised of covalently linked subunits callednucleotides. Nucleic acid includes polyribonucleic acid (RNA) andpolydeoxyribonucleic acid (DNA), both of which may be single-stranded ordouble-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, andsemi-synthetic DNA.

An “isolated nucleic acid molecule” or “isolated nucleic acid fragment”refers to the phosphate ester polymeric form of ribonucleosides(adenosine, guanosine, uridine or cytidine; “RNA molecules”) ordeoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, ordeoxycytidine; “DNA molecules”), or any phosphoester analogs thereof,such as phosphorothioates and thioesters, in either single strandedform, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA andRNA-RNA helices are possible. The term nucleic acid molecule, and inparticular DNA or RNA molecule, refers only to the primary and secondarystructure of the molecule, and does not limit it to any particulartertiary forms. Thus, this term includes double-stranded DNA found,inter alia, in linear or circular DNA molecules (e.g., restrictionfragments), plasmids, and chromosomes. In discussing the structure ofparticular double-stranded DNA molecules, sequences may be describedherein according to the normal convention of giving only the sequence inthe 5′ to 3′ direction along the non-transcribed strand of DNA (i.e.,the strand having a sequence homologous to the mRNA).

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein, includingintervening sequences (introns) between individual coding segments(exons), as well as regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified, e.g., in Sambrook, J., Fritsch, E. F. and Maniatis, T.MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter11 and Table 11.1 therein (hereinafter “Maniatis”, entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments, such ashomologous sequences from distantly related organisms, to highly similarfragments, such as genes that duplicate functional enzymes from closelyrelated organisms. Post-hybridization washes determine stringencyconditions. One set of conditions uses a series of washes starting with6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 min. For more stringent conditions,washes are performed at higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS are increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C. An additional set of highly stringent conditions are definedby hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC,0.1% SDS followed by 0.1×SSC, 0.1% SDS.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see, e.g.,Maniatis at 9.50-9.51). For hybridizations with shorter nucleic acids,i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see, e.g., Maniatis, at 11.7-11.8). In one embodiment thelength for a hybridizable nucleic acid is at least about 10 nucleotides.Preferably a minimum length for a hybridizable nucleic acid is at leastabout 15 nucleotides; more preferably at least about 20 nucleotides; andmost preferably the length is at least 30 nucleotides. Furthermore, theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as length of the probe.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences.

As known in the art, “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

“Identity” and “similarity” can be readily calculated by known methods,including but not limited to those described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, NY (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994);Sequence Analysis in Molecular Biology (von Heinje, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Stockton Press, NY (1991). Preferred methods to determineidentity are designed to give the best match between the sequencestested. Methods to determine identity and similarity are codified inpublicly available computer programs. Sequence alignments and percentidentity calculations may be performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignments of the sequences disclosed herein were performedusing the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10). Default parameters for pairwise alignments using theClustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5.

Suitable nucleic acid sequences or fragments thereof (isolatedpolynucleotides of the present invention) encode polypeptides that areat least about 70% to 75% identical to the amino acid sequences reportedherein, at least about 80%, 85%, or 90% identical to the amino acidsequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequences reported herein. Suitablenucleic acid fragments are at least about 70%, 75%, or 80% identical tothe nucleic acid sequences reported herein, at least about 80%, 85%, or90% identical to the nucleic acid sequences reported herein, or at leastabout 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acidsequences reported herein. Suitable nucleic acid fragments not only havethe above identities/similarities but typically encode a polypeptidehaving at least 50 amino acids, at least 100 amino acids, at least 150amino acids, at least 200 amino acids, or at least 250 amino acids.

A DNA or RNA “coding region” is a DNA or RNA molecule which istranscribed and/or translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. “Suitable regulatory regions” refer to nucleic acid regionslocated upstream (5′ non-coding sequences), within, or downstream (3′non-coding sequences) of a coding region, and which influence thetranscription, RNA processing or stability, or translation of theassociated coding region. Regulatory regions may include promoters,translation leader sequences, RNA processing site, effector binding siteand stem-loop structure. The boundaries of the coding region aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxyl) terminus A coding region can include,but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNAmolecules, synthetic DNA molecules, or RNA molecules. If the codingregion is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding region.

An “isoform” is a protein that has the same function as another proteinbut which is encoded by a different gene and may have small differencesin its sequence.

A “paralogue” is a protein encoded by a gene related by duplicationwithin a genome.

An “orthologue” is gene from a different species that has evolved from acommon ancestral gene by speciation. Normally, orthologues retain thesame function in the course of evolution as the ancestral gene.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid, either DNA, cDNA or RNA, that comprises a translation start signalor initiation codon, such as an ATG or AUG, and a termination codon andcan be potentially translated into a polypeptide sequence.

“Promoter” refers to a DNA fragment capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingregion is located 3′ to a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity. A promoter isgenerally bounded at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter will be found atranscription initiation site (conveniently defined for example, bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

A coding region is “under the control” of transcriptional andtranslational control elements in a cell when RNA polymerase transcribesthe coding region into mRNA, which is then trans-RNA spliced (if thecoding region contains introns) and translated into the protein encodedby the coding region.

“Transcriptional and translational control regions” are DNA regulatoryregions, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding region in a host cell. Ineukaryotic cells, polyadenylation signals are control regions.

The term “operably associated” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably associatedwith a coding region when it is capable of affecting the expression ofthat coding region (i.e., that the coding region is under thetranscriptional control of the promoter). Coding regions can be operablyassociated to regulatory regions in sense or antisense orientation.

The term “expression,” as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

The term “lignocellulose” refers to material that is comprised of ligninand cellulose.

A “cellulolytic enzyme” can be any enzyme involved in cellulosedigestion, metabolism and/or hydrolysis. The term “cellulase” refers toa class of enzymes produced chiefly by fungi, bacteria, and protozoansthat catalyze cellulolysis (i.e. the hydrolysis) of cellulose. However,there are also cellulases produced by other types of organisms such asplants and animals. Several different kinds of cellulases are known,which differ structurally and mechanistically. There are general typesof cellulases based on the type of reaction catalyzed: endocellulasebreaks internal bonds to disrupt the crystalline structure of celluloseand expose individual cellulose polysaccharide chains; exocellulasecleaves 2-4 units from the ends of the exposed chains produced byendocellulase, resulting in the tetrasaccharides or disaccharide such ascellobiose. There are two main types of exocellulases (orcellobiohydrolases, abbreviate CBH)—one type working processively fromthe reducing end, and one type working processively from thenon-reducing end of cellulose; cellobiase or beta-glucosidase hydrolysesthe exocellulase product into individual monosaccharides; oxidativecellulases that depolymerize cellulose by radical reactions, as forinstance cellobiose dehydrogenase (acceptor); cellulose phosphorylasesthat depolymerize cellulose using phosphates instead of water. In themost familiar case of cellulase activity, the enzyme complex breaks downcellulose to beta-glucose. A “cellulase” can be any enzyme involved incellulose digestion, metabolism and/or hydrolysis, including anendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, and feruoyl esterase protein.

An “amylolytic enzyme” can be any enzyme involved in amylase digestion,metabolism and/or hydrolysis. The term “amylase” refers to an enzymethat breaks starch down into sugar. Amylase is present in human saliva,where it begins the chemical process of digestion. Foods that containmuch starch but little sugar, such as rice and potato, taste slightlysweet as they are chewed because amylase turns some of their starch intosugar in the mouth. The pancreas also makes amylase (α-amylase) tohydrolyse dietary starch into disaccharides and trisaccharides which areconverted by other enzymes to glucose to supply the body with energy.Plants and some bacteria also produce amylase. All amylases areglycoside hydrolases and act on α-1,4-glycosidic bonds. Some amylases,such as γ-amylase (glucoamylase), also act on α-1,6-glycosidic bonds.Amylase enzymes include α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2),and γ-amylase (EC 3.2.1.3). The α-amylases are calcium metalloenzymes,unable to function in the absence of calcium. By acting at randomlocations along the starch chain, α-amylase breaks down long-chaincarbohydrates, ultimately yielding maltotriose and maltose from amylose,or maltose, glucose and “limit dextrin” from amylopectin. Because it canact anywhere on the substrate, α-amylase tends to be faster-acting thanβ-amylase. In animals, it is a major digestive enzyme and its optimum pHis about 6.7-7.0. Another form of amylase, β-amylase is also synthesizedby bacteria, fungi, and plants. Working from the non-reducing end,β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond,cleaving off two glucose units (maltose) at a time. Many microbesproduce amylase to degrade extracellular starches. In addition tocleaving the last α(1-4)glycosidic linkages at the nonreducing end ofamylose and amylopectin, yielding glucose, γ-amylase will cleave α(1-6)glycosidic linkages. Another amylolytic enzyme is alpha-glucosidase thatacts on maltose and other short malto-oligosaccharides produced byalpha-, beta-, and gamma-amylases, converting them to glucose. Anotheramylolytic enzyme is pullulanase. Pullulanase is a specific kind ofglucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan isregarded as a chain of maltotriose units linked by alpha-1,6-glycosidicbonds. Pullulanase (EC 3.2.1.41) is also known aspullulan-6-glucanohydrolase (Debranching enzyme). Another amylolyticenzyme, isopullulanase, hydrolyses pullulan to isopanose(6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1.57) is also known aspullulan 4-glucanohydrolase. An “amylase” can be any enzyme involved inamylase digestion, metabolism and/or hydrolysis, including α-amylase,β-amylase, glucoamylase, pullulanase, isopullulanase, andalpha-glucosidase.

The term “xylanolytic activity” is intended to include the ability tohydrolyze glycosidic linkages in oligopentoses and polypentoses. Theterm “xylanase” is the name given to a class of enzymes which degradethe linear polysaccharide beta-1,4-xylan into xylose, thus breaking downhemicellulose, one of the major components of plant cell walls. As such,it plays a major role in micro-organisms thriving on plant sources(mammals, conversely, do not produce xylanase). Additionally, xylanasesare present in fungi for the degradation of plant matter into usablenutrients. Xylanases include those enzymes that correspond to EnzymeCommission Number 3.2.1.8. A “xylose metabolizing enzyme” can be anyenzyme involved in xylose digestion, metabolism and/or hydrolysis,including a xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and a xylose transaldolase protein.

The term “pectinase” is a general term for enzymes, such as pectolyase,pectozyme and polygalacturonase, commonly referred to in brewing aspectic enzymes. These enzymes break down pectin, a polysaccharidesubstrate that is found in the cell walls of plants. One of the moststudied and widely used commercial pectinases is polygalacturonase.Pectinases are commonly used in processes involving the degradation ofplant materials, such as speeding up the extraction of fruit juice fromfruit, including apples and sapota. Pectinases have also been used inwine production since the 1960s.

A “saccharolytic enzyme” can be any enzyme involved in carbohydratedigestion, metabolism and/or hydrolysis, including amylases, cellulases,hemicellulases, cellulolytic and amylolytic accessory enzymes,inulinases, levanases, and pentose sugar utilizing enzymes.

A “pentose sugar utilizing enzyme” can be any enzyme involved in pentosesugar digestion, metabolism and/or hydrolysis, including xylanase,arabinase, arabinoxylanase, arabinosidase, arabinofuranosidase,arabinoxylanase, arabinosidase, and arabinofuranosidase, arabinoseisomerase, ribulose-5-phosphate 4-epimerase, xylose isomerase,xylulokinase, xylose reductase, xylose dehydrogenase, xylitoldehydrogenase, xylonate dehydratase, xylose transketolase, and/or xylosetransaldolase.

Host Cells Expressing Heterologous Saccharolytic Enzymes

In order to address the limitations of the previous systems, in oneaspect, the present invention provides host cells expressingheterologous cellulases that can be effectively and efficiently utilizedto produce products such as ethanol from cellulose. In anotherembodiment, the host cells express heterologous amylases that can beeffectively and efficiently utilized to produce products such as ethanolfrom biomass feedstock, such as grain feedstock. In yet anotherembodiment, the host cells express heterologous enzymes that utilizepentose sugars.

In some embodiments, the host cell can be a yeast. According to thepresent invention the yeast host cell can be, for example, from thegenera Saccharomyces, Kluyveromyces, Candida, Pichia,Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia.Yeast species as host cells can include, for example, S. cerevisiae, S.bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis,K marxianus, or K. fragilis. In some embodiments, the yeast is selectedfrom the group consisting of Saccharomyces cerevisiae,Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichiastipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma,Candida utilis, Arxula adeninivorans, Debaryomyces hansenii,Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomycesoccidentalis. In one particular embodiment, the yeast is Saccharomycescerevisiae. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

In some embodiments of the present invention, the host cell is anoleaginous cell. According to the present invention, the oleaginous hostcell can be an oleaginous yeast cell. For example, the oleaginous yeasthost cell can be from the genera Blakeslea, Candida, Cryptococcus,Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium,Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. According to thepresent invention, the oleaginous host cell can be an oleaginousmicroalgae host cell. For example, the oleaginous microalgea host cellcan be from the genera Thraustochytrium or Schizochytrium.

In some embodiments of the present invention, the host cell is athermotolerant host cell. Thermotolerant host cells can be particularlyuseful in simultaneous saccharification and fermentation processes byallowing externally produced cellulases and ethanol-producing host cellsto perform optimally in similar temperature ranges.

Thermotolerant host cells of the invention can include, for example,Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana,Pichia farinosa, Clavispora opuntiae, Clavispora lusitaniae, Candidamexicana, Hansenula polymorpha and Kluyveromyces host cells.

In some particular embodiments of the present invention, the host cellis a Kluyveromyces host cell. For example, the Kluyveromyces host cellcan be a K. lactis, K. marxianus, K. blattae, K. phaffii, K. yarrowii,K. aestuarii, K. dobzhanskii, K. wickerhamii, K. thermotolerans, or K.waltii host cell. In one embodiment, the host cell is a K. lactis, or K.marxianus host cell. In another embodiment, the host cell is a K.marxianus host cell.

In some embodiments of the present invention the thermotolerant hostcell can grow at temperatures above about 30° C., about 31° C., about32° C., about 33° C., about 34° C., about 35° C., about 36° C., about37° C., about 38° C., about 39° C., about 40° C., about 41° C. or about42° C. In some embodiments of the present invention the thermotoleranthost cell can produce ethanol from cellulose at temperatures above about30° C., about 31° C., about 32° C., about 33° C., about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., about40° C., about 41° C., about 42° C., or about 50° C.

In some embodiments of the present invention, the thermotolerant hostcell can grow at temperatures from about 30° C. to 60° C., about 30° C.to 55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C.to 55° C. or about 40° C. to 50° C. In some embodiments of the presentinvention, the thermotolterant host cell can produce ethanol fromcellulose at temperatures from about 30° C. to 60° C., about 30° C. to55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C. to55° C. or about 40° C. to 50° C.

Host cells are genetically engineered (transduced or transformed ortransfected) with the polynucleotides encoding saccharolytic enzymes(amylases, cellulases, hemicellulases, cellulolytic and amylolyticaccessory enzymes, inulinases, levanases, pentose sugar hydrolases andothers) of this invention which are described in more detail herein. Thepolynucleotides encoding saccharolytic enzymes can be introduced to thehost cell on a vector of the invention, which may be, for example, acloning vector or an expression vector comprising a sequence encoding aheterologous saccharolytic enzyme. The host cells can comprisepolynucleotides of the invention as integrated copies or plasmid copies.

In certain aspects, the present invention relates to host cellscontaining the polynucleotide constructs described herein. In oneembodiment, the host cells of the present invention express one or moreheterologous polypeptides of saccharolytic enzymes. In some embodiments,the host cell comprises a combination of polynucleotides that encodeheterologous saccharolytic enzymes or fragments, variants or derivativesthereof. The host cell can, for example, comprise multiple copies of thesame nucleic acid sequence, for example, to increase expression levels,or the host cell can comprise a combination of unique polynucleotides.In other embodiments, the host cell comprises a single polynucleotidethat encodes a heterologous saccharolytic enzyme or a fragment, variantor derivative thereof. In particular, such host cells expressing asingle heterologous saccharolytic enzyme can be used in co-culture withother host cells of the invention comprising a polynucleotide thatencodes at least one other heterologous saccharolytic enzyme orfragment, variant or derivative thereof.

Introduction of a polynucleotide encoding a heterologous saccharolyticenzyme into a host cell can be done by methods known in the art.Introduction of polynucleotides encoding heterologous saccharolyticenzyme into, for example yeast host cells, can be effected by lithiumacetate transformation, spheroplast transformation, or transformation byelectroporation, as described in Current Protocols in Molecular Biology,13.7.1-13.7.10. Introduction of the construct in other host cells can beeffected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., et al., Basic Methods inMolecular Biology, (1986)).

The transformed host cells or cell cultures, as described above, can beexamined for protein content of an endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase protein, alpha-amylase, beta-amylase,glucoamylase, pullulanase, isopullulanase, alpha-glucosidase,beta-glucosidase, arabinase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase. For the use of secretedheterologous saccharolytic enzymes, protein content can be determined byanalyzing the host (e.g., yeast) cell supernatants. In certainembodiments, high molecular weight material can be recovered from theyeast cell supernatant either by acetone precipitation or by bufferingthe samples with disposable de-salting cartridges. Proteins, includingtethered heterologous saccharolytic enzymes, can also be recovered andpurified from recombinant yeast cell cultures by methods includingspheroplast preparation and lysis, cell disruption using glass beads,and cell disruption using liquid nitrogen for example. Additionalprotein purification methods include ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, gel filtration,and lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Protein analysis methods include methods such as the traditional Lowrymethod, the BCA assay, absorbance at 280 nm, or the protein assay methodaccording to BioRad's manufacturer's protocol. Using such methods, theprotein content of saccharolytic enzymes can be estimated. Additionally,to accurately measure protein concentration a heterologous cellulase canbe expressed with a tag, for example a His-tag or HA-tag and purified bystandard methods using, for example, antibodies against the tag, astandard nickel resin purification technique or similar approach.

The transformed host cells or cell cultures, as described above, can befurther analyzed for hydrolysis of cellulose, or starch, or pentosesugar utilization (e.g., by a sugar detection assay), for a particulartype of saccharolytic enzyme activity (e.g., by measuring the individualendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, and arabinofuranosidase, arabinose isomerase,ribulose-5-phosphate 4-epimerase, xylose isomerase, xylulokinase, xylosereductase, xylose dehydrogenase, xylitol dehydrogenase, xylonatedehydratase, xylose transketolase, and/or xylose transaldolase) or fortotal cellulase activity. Endoglucanase activity can be determined, forexample, by measuring an increase of reducing ends in an endoglucanasespecific CMC or hydroxyethylcellulose (HEC) substrate. Cellobiohydrolaseactivity can be measured, for example, by using insoluble cellulosicsubstrates such as the amorphous substrate phosphoric acid swollencellulose (PASC) or microcrystalline cellulose (Avicel) and determiningthe extent of the substrate's hydrolysis. β-glucosidase activity can bemeasured by a variety of assays, e.g., using cellobiose. Assays foractivity of other saccharolytic enzyme types are known in the art andare exemplified below.

A total saccharolytic enzyme activity, which can include the activity ofendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase protein, alpha-amylase,beta-amylase, glucoamylase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, pullulanase,isopullulanase, arabinose isomerase, ribulose-5-phosphate 4-epimerase,xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase,xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, andxylose transaldolase can hydrolyze biomass feedstocks synergistically.For example, total cellulase activity can thus be measured usinginsoluble substrates including pure cellulosic substrates such asWhatman No. 1 filter paper, cotton linter, microcrystalline cellulose,bacterial cellulose, algal cellulose, and cellulose-containingsubstrates such as dyed cellulose, alpha-cellulose or pretreatedlignocellulose. Specific activity of cellulases can also be detected bymethods known to one of ordinary skill in the art, such as by the Avicelassay (described supra) that would be normalized by protein (cellulase)concentration measured for the sample. Total saccharolytic activitycould be also measured using complex substrate containing starch,cellulose and hemicellulose such as corn mash by measuring releasedmonomeric sugars. In such an assay different groups of enzymes couldwork in “indirect” when one group of enzymes such as cellulases can makesubstrate for another group of enzymes such as amylases more accessiblethrough hydrolysis of cellulolytic substrate around amylolyticsubstrate. This mechanism can also work vice versa.

One aspect of the invention is thus related to the efficient productionof saccharolytic enzymes to aid in the digestion and utilization ofstarch, cellulose, and pentose sugars, and generation of products suchas ethanol. A “saccharolytic enzyme” can be any enzyme involved incarbohydrate digestion, metabolism and/or hydrolysis, includingamylases, cellulases, hemicellulases, cellulolytic and amylolyticaccessory enzymes, inulinases, levanases, and pentose sugar hydrolasingenzymes. A “cellulase” can be any enzyme involved in cellulasedigestion, metabolism and/or hydrolysis, including an endoglucanase,glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylanesterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, and feruoyl esterase protein. An “amylase” can be any enzymeinvolved in amylase digestion and/or metabolism, includingalpha-amylase, beta-amylase, glucoamylase, pullulanase, isopullulanase,and alpha-glucosidase. A pentose sugar hydrolyzing enzyme can be anyenzyme involved in pentose sugar digestion, and/or metabolism, includingxylanase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase.

In additional embodiments, the transformed host cells or cell culturesare assayed for ethanol production. Ethanol production can be measuredby techniques known to one or ordinary skill in the art, e.g., by astandard HPLC refractive index method.

Heterologous Saccharolytic Enzymes

According to one aspect of the present invention, the expression ofheterologous saccharolytic enzymes in a host cell can be usedadvantageously to produce products such as ethanol from biomass sources.For example, cellulases from a variety of sources can be heterologouslyexpressed to successfully increase efficiency of ethanol production. Thesaccharolytic enzymes can be from fungi, yeast, bacteria, plant,protozoan or termite sources. In some embodiments, the saccharolyticenzyme is from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C.lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M.darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus,Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum,Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagusdegradans, Piromyces equii, Neocallimastix patricarum or Arabidopsisthaliana.

In some embodiments, the cellulase of the invention is any cellulasedisclosed in Table 4 or Table 7 produced herein. In some embodiments,the cellulase is encoded by a nucleic acid sequence at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% identical to any one of SEQ ID NOs: 1-218. In some embodiments,the cellulase has an amino acid sequence that is at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%identical to any one of SEQ ID NOs: 219-436. In some embodiments, thecellulase of the invention is any cellulase suitable for expression inan appropriate host cell.

In other embodiments, the amylase of the invention is any amylasedisclosed in Table 19 produced herein. In some embodiments, the amylaseis encoded by a nucleic acid sequence at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%identical to any one of SEQ ID NOs: 437-441. In some embodiments, thecellulase has an amino acid sequence that is at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%identical to any one of SEQ ID NOs: 442-446. In some embodiments, theamylase of the invention is any amylase suitable for expression in anappropriate host cell.

In some embodiments of the invention, multiple saccharolytic enzymesfrom a single organism are co-expressed in the same host cell. In someembodiments of the invention, multiple saccharolytic enzymes fromdifferent organisms are co-expressed in the same host cell. Inparticular, saccharolytic enzymes from two, three, four, five, six,seven, eight, nine or more organisms can be co-expressed in the samehost cell. Similarly, the invention can encompass co-cultures of yeaststrains, wherein the yeast strains express different saccharolyticenzymes. Co-cultures can include yeast strains expressing heterologoussaccharolytic enzymes from the same organisms or from differentorganisms. Co-cultures can include yeast strains expressingsaccharolytic enzymes from two, three, four, five, six, seven, eight,nine or more organisms.

Lignocellulases of the present invention include both endoglucanases andexoglucanases. Other lignocellulases of the invention include accesoryenzymes which can act on the lignocellulosic material. Thelignocellulases can be, for example, endoglucanases, glucosidases,cellobiohydrolases, xylanases, glucanases, xylosidases, xylan esterases,arabinofuranosidases, galactosidases, cellobiose phosphorylases,cellodextrin phosphorylases, mannanases, mannosidases, xyloglucanases,endoxylanases, glucuronidases, acetylxylanesterases,arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins,pectinases, and feruoyl esterases. In some embodiments, thelignocellulases of the invention can be any suitable enzyme fordigesting the desired lignocellulosic material.

In certain embodiments of the invention, the lignocellulase can be anendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, and feruoyl esterase paralogue ororthologue. In particular embodiments, the lignocellulase is derivedfrom any species named in Tables 4 and 7. In one particular embodiment,the lignocellulase comprises an amino acid sequence selected from SEQ IDNOs: 219-436. In certain other embodiments, the lignocellulase comprisesan amino acid sequence that is at least about 70, about 80, about 90,about 95, about 96, about 97, about 98, about 99, or 100% identical toan amino acid sequence selected from SEQ ID NOs: 219-436.

In other embodiments of the invention, the amylases can bealpha-amylases, beta-amylases, glucoamylases, alpha-glucosidases,pullulanase, or isopullulanase paralogues or orthologues.

As a practical matter, whether any polypeptide is at least 70%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide ofthe present invention can be determined conventionally using knowncomputer programs. Methods for determining percent identity, asdiscussed in more detail below in relation to polynucleotide identity,are also relevant for evaluating polypeptide sequence identity.

In some particular embodiments of the invention, the saccharolyticenzyme comprises a sequence selected from the saccharolytic enzymesdisclosed in Table 4, or Table 7, or Table 19 presented herein. Thesaccharolytic enzymes of the invention also include saccharolyticenzymes that comprise a sequence at least about 70, about 80, about 90,about 95, about 96, about 97, about 98, about 99 or 100% identical tothe sequences of Table 4, or Table 7, or Table 19. Amino acid andnucleic acid sequences are readily determined for a gene, protein orother element by a accession number upon consulting the proper database,for example Genebank. However, sequences for the genes and proteins ofthe present invention are also disclosed herein (SEQ ID NOs: 1-445).

Some embodiments of the invention encompass a polypeptide comprising atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 ormore consecutive amino acids of any of SEQ ID NOs: 219-445, or domains,fragments, variants, or derivatives.

In certain aspects of the invention, the polypeptides andpolynucleotides of the present invention are provided in an isolatedform, e.g., purified to homogeneity.

The present invention also encompasses polypeptides which comprise, oralternatively consist of, an amino acid sequence which is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% similar to the polypeptide of anyof SEQ ID NOs: 219-436, or SEQ ID NOs:442-446, and to portions of suchpolypeptide with such portion of the polypeptide generally containing atleast 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

The present invention further relates to a domain, fragment, variant,derivative, or analog of the polypeptide of any of SEQ ID NOs: 219-436,or SEQ ID NOs:442-446.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis. Therefore, the fragments can be employed asintermediates for producing the full-length polypeptides.

Fragments of lignocellulases of the invention encompass domains,proteolytic fragments, deletion fragments and in particular, fragmentsof any of the genes named in Tables 4 and 7, which retain any specificbiological activity of the endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, and feruoyl esterase proteins. Polypeptide fragments furtherinclude any portion of the polypeptide which retains a catalyticactivity of endoglucanase, glucosidase, cellobiohydrolase, xylanase,glucanase, xylosidase, xylan esterase, arabinofuranosidase,galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase,mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, and feruoyl esterase protein.

Fragments of amylases of the invention encompass domains, proteolyticfragments, deletion fragments and in particular, fragments of any of thegenes named in Tables 15, 16, and 19, which retain any specificbiological activity of the alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, and alpha-glucosidase proteins. Polypeptidefragments further include any portion of the polypeptide which retains acatalytic activity of alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, and alpha-glucosidase protein.

The variant, derivative or analog of the polypeptide of any of SEQ IDNOs: 219-436, or SEQ ID NOs:442-446 may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide for purification of the polypeptideor (v) one in which a fragment of the polypeptide is soluble, i.e., notmembrane bound, yet still binds ligands to the membrane bound receptor.Such variants, derivatives and analogs are deemed to be within the scopeof those skilled in the art from the teachings herein.

The polypeptides of the present invention further include variants ofthe polypeptides. A “variant” of the polypeptide can be a conservativevariant, or an allelic variant. As used herein, a conservative variantrefers to alterations in the amino acid sequence that do not adverselyaffect the biological functions of the protein. A substitution,insertion or deletion is said to adversely affect the protein when thealtered sequence prevents or disrupts a biological function associatedwith the protein. For example, the overall charge, structure orhydrophobic-hydrophilic properties of the protein can be altered withoutadversely affecting a biological activity. Accordingly, the amino acidsequence can be altered, for example to render the peptide morehydrophobic or hydrophilic, without adversely affecting the biologicalactivities of the protein.

By an “allelic variant” is intended alternate forms of a gene occupyinga given locus on a chromosome of an organism. Genes II, Lewin, B., ed.,John Wiley & Sons, New York (1985). Non-naturally occurring variants maybe produced using art-known mutagenesis techniques. Allelic variants,though possessing a slightly different amino acid sequence than thoserecited above, will still have the same or similar biological functionsassociated with the endoglucanases, glucosidases, cellobiohydrolases,xylanases, glucanases, xylosidases, xylan esterases,arabinofuranosidases, galactosidases, cellobiose phosphorylases,cellodextrin phosphorylases, mannanases, mannosidases, xyloglucanases,endoxylanases, glucuronidases, acetylxylanesterases,arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins,pectinases, feruoyl esterases, alpha-amylase, beta-amylase,glucoamylase, alpha-glucosidase, beta-glucosidase, galactosidase,arabinase, arabinoxylanase, arabinosidase, arabinofuranosidase,arabinoxylanase, arabinosidase, and arabinofuranosidase, arabinoseisomerase, ribulose-5-phosphate 4-epimerase, xylose isomerase,xylulokinase, xylose reductase, xylose dehydrogenase, xylitoldehydrogenase, xylonate dehydratase, xylose transketolase, and/or xylosetransaldolase of the invention. The allelic variants, the conservativesubstitution variants, and members of the endoglucanase,cellobiohydrolase, 3-glucosidase, alpha-amylase, beta-amylase,glucoamylase, pullulanase, isopullulanase, or alpha-glucosidase proteinfamilies, can have an amino acid sequence having at least 75%, at least80%, at least 90%, at least 95% amino acid sequence identity withendoglucanases, glucosidases, cellobiohydrolases, xylanases, glucanases,xylosidases, xylan esterases, arabinofuranosidases, galactosidases,cellobiose phosphorylases, cellodextrin phosphorylases, mannanases,mannosidases, xyloglucanases, endoxylanases, glucuronidases,acetylxylanesterases, arabinofuranohydrolases, swollenins, glucuronylesterases, expansins, pectinases, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, and beta-glucosidase amino acid sequence set forth inany one of SEQ ID NOs: 219-436, and SEQ ID NOs: 442-446. Identity orhomology with respect to such sequences is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the known peptides, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. N-terminal, C-terminal or internal extensions,deletions, or insertions into the peptide sequence shall not beconstrued as affecting homology.

Thus, in one aspect the proteins and peptides of the present inventioninclude molecules comprising the amino acid sequence of SEQ ID NOs:219-436, or and SEQ ID NOs: 442-446 or fragments thereof having aconsecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30,35 or more amino acid residues of the endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, and beta-glucosidasepolypeptide sequences; amino acid sequence variants of such sequenceswherein at least one amino acid residue has been inserted N- orC-terminal to, or within, the disclosed sequence; amino acid sequencevariants of the disclosed sequences, or their fragments as definedabove, that have been substituted by another residue. Contemplatedvariants further include those containing predetermined mutations by,e.g., homologous recombination, site-directed or PCR mutagenesis, andthe corresponding proteins of other animal species, including but notlimited to bacterial, fungal, insect, rabbit, rat, porcine, bovine,ovine, equine and non-human primate species, the alleles or othernaturally occurring variants of the family of proteins; and derivativeswherein the protein has been covalently modified by substitution,chemical, enzymatic, or other appropriate means with a moiety other thana naturally occurring amino acid (for example, a detectable moiety suchas an enzyme or radioisotope).

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of saccharolytic enzymes. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the secreted protein without substantial loss ofbiological function.

Thus, in another aspect the invention further includes endoglucanase,glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylanesterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and xylose transaldolase polypeptide variants which showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

The skilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare often surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

The terms “derivative” and “analog” refer to a polypeptide differingfrom the endoglucanases, glucosidases, cellobiohydrolases, xylanases,glucanases, xylosidases, xylan esterases, arabinofuranosidases,galactosidases, cellobiose phosphorylases, cellodextrin phosphorylases,mannanases, mannosidases, xyloglucanases, endoxylanases, glucuronidases,acetylxylanesterases, arabinofuranohydrolases, swollenins, glucuronylesterases, expansins, pectinases, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, and arabinofuranosidase, arabinose isomerase,ribulose-5-phosphate 4-epimerase, xylose isomerase, xylulokinase, xylosereductase, xylose dehydrogenase, xylitol dehydrogenase, xylonatedehydratase, xylose transketolase, and xylose transaldolase polypeptidesas disclosed herein, but retaining essential properties thereof.Generally, derivatives and analogs are overall closely similar, and, inmany regions, identical to the endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and xylose transaldolase polypeptides disclosed herein.The terms “derivative” and “analog” when referring to endoglucanases,glucosidases, cellobiohydrolases, xylanases, glucanases, xylosidases,xylan esterases, arabinofuranosidases, galactosidases, cellobiosephosphorylases, cellodextrin phosphorylases, mannanases, mannosidases,xyloglucanases, endoxylanases, glucuronidases, acetylxylanesterases,arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins,pectinases, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and xylose transaldolase polypeptides include anypolypeptides which retain at least some of the activity of thecorresponding native polypeptide, e.g., the exoglucanase activity, orthe activity of the its catalytic domain.

Derivatives of the saccharolytic enzymes disclosed herein, arepolypeptides which have been altered so as to exhibit features not foundon the native polypeptide. Derivatives can be covalently modified bysubstitution, chemical, enzymatic, or other appropriate means with amoiety other than a naturally occurring amino acid (for example, adetectable moiety such as an enzyme or radioisotope). Examples ofderivatives include fusion proteins.

An analog is another form of an endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and xylose transaldolase polypeptide of the presentinvention. An “analog” also retains substantially the same biologicalfunction or activity as the polypeptide of interest, e.g., functions asa xylanase. An analog includes a proprotein which can be activated bycleavage of the proprotein portion to produce an active maturepolypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. In someparticular embodiments, the polypeptide is a recombinant polypeptide.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to any of SEQ ID NOs: 1-218, or SEQ ID NOs: 437-441 usinginformation from the sequences disclosed herein or the clones depositedwith the ATCC. For example, allelic variants and/or species homologs maybe isolated and identified by making suitable probes or primers from thesequences provided herein and screening a suitable nucleic acid sourcefor allelic variants and/or the desired homologue.

Combinations of Saccharolytic Enzymes

In some embodiments of the present invention, the host cell expresses acombination of heterologous saccharolytic enzymes. For example, the hostcell can contain at least two heterologous saccharolytic enzymes, atleast three heterologous saccharolytic enzymes, at least fourheterologous saccharolytic enzymes, at least five heterologoussaccharolytic enzymes, at least six heterologous saccharolytic enzymes,at least seven heterologous saccharolytic enzymes, at least eightheterologous saccharolytic enzymes, at least nine heterologoussaccharolytic enzymes, at least ten heterologous saccharolytic enzymes,at least eleven heterologous saccharolytic enzymes, at least twelveheterologous saccharolytic enzymes, at least thirteen heterologoussaccharolytic enzymes, at least fourteen heterologous saccharolyticenzymes, or at least fifteen heterologous saccharolytic enzymes. Theheterologous saccharolytic enzymes in the host cell can be from the sameor from different species. In one embodiment the host cell expressesheterologous enzymes comprising cellobiohydrolases, endo-gluconases,beta-glucosidases, xylanases, xylosidases, glucoamylases,alpha-amylases, alpha-glucosidases, pullulanases, isopullulanases,pectinases, and acetylxylan esterases.

Tethered and Secreted Saccharolytic Enzymes

According to the present invention, the saccharolytic enzymes can beeither tethered or secreted. As used herein, a protein is “tethered” toan organism's cell surface if at least one terminus of the protein isbound, covalently and/or electrostatically for example, to the cellmembrane or cell wall. It will be appreciated that a tethered proteincan include one or more enzymatic regions that can be joined to one ormore other types of regions at the nucleic acid and/or protein levels(e.g., a promoter, a terminator, an anchoring domain, a linker, asignaling region, etc.). While the one or more enzymatic regions may notbe directly bound to the cell membrane or cell wall (e.g., such as whenbinding occurs via an anchoring domain), the protein is nonethelessconsidered a “tethered enzyme” according to the present specification.

Tethering can, for example, be accomplished by incorporation of ananchoring domain into a recombinant protein that is heterologouslyexpressed by a cell, or by prenylation, fatty acyl linkage, glycosylphosphatidyl inositol anchors or other suitable molecular anchors whichmay anchor the tethered protein to the cell membrane or cell wall of thehost cell. A tethered protein can be tethered at its amino terminal endor optionally at its carboxy terminal end.

As used herein, “secreted” means released into the extracellular milieu,for example into the media. Although tethered proteins may havesecretion signals as part of their immature amino acid sequence, theyare maintained as attached to the cell surface, and do not fall withinthe scope of secreted proteins as used herein.

As used herein, “flexible linker sequence” refers to an amino acidsequence which links two amino acid sequences, for example, a cell wallanchoring amino acid sequence with an amino acid sequence that containsthe desired enzymatic activity. The flexible linker sequence allows fornecessary freedom for the amino acid sequence that contains the desiredenzymatic activity to have reduced steric hindrance with respect toproximity to the cell and may also facilitate proper folding of theamino acid sequence that contains the desired enzymatic activity.

In some embodiments of the present invention, the tethered cellulaseenzymes are tethered by a flexible linker sequence linked to ananchoring domain. In some embodiments, the anchoring domain is of CWP2(for carboxy terminal anchoring) or FLO1 (for amino terminal anchoring)from S. cerevisiae.

In some embodiments, heterologous secretion signals may be added to theexpression vectors of the present invention to facilitate theextra-cellular expression of cellulase proteins. In some embodiments,the heterologous secretion signal is the secretion signal from T. reeseiXyn2. In other embodiments, the heterologous secretion signal is the S.cerevisiae Invertase signal. In yet other embodiments, the heterologoussecretion signal is the S. cerevisiae AF mating signal.

Fusion Proteins Comprising Saccharolytic Enzymes

The present invention also encompasses fusion proteins. For example, thefusion proteins can be a fusion of a heterologous saccharolytic enzymeand a second peptide. The heterologous saccharolytic enzyme and thesecond peptide can be fused directly or indirectly, for example, througha linker sequence. The fusion protein can comprise for example, a secondpeptide that is N-terminal to the heterologous saccharolytic enzymeand/or a second peptide that is C-terminal to the heterologoussaccharolytic enzyme. Thus, in certain embodiments, the polypeptide ofthe present invention comprises a first polypeptide and a secondpolypeptide, wherein the first polypeptide comprises a heterologoussaccharolytic enzyme.

According to one aspect of the present invention, the fusion protein cancomprise a first and second polypeptide wherein the first polypeptidecomprises a heterologous saccharolytic enzyme and the second polypeptidecomprises a signal sequence. According to another embodiment, the fusionprotein can comprise a first and second polypeptide, wherein the firstpolypeptide comprises a heterologous saccharolytic enzyme and the secondpolypeptide comprises a polypeptide used to facilitate purification oridentification or a reporter peptide. The polypeptide used to facilitatepurification or identification or the reporter peptide can be, forexample, a HIS-tag, a GST-tag, an HA-tag, a FLAG-tag, a MYC-tag, or afluorescent protein.

According to yet another embodiment, the fusion protein can comprise afirst and second polypeptide, wherein the first polypeptide comprises aheterologous saccharolytic enzyme and the second polypeptide comprisesan anchoring peptide. In some embodiments, the anchoring domain is ofCWP2 (for carboxy terminal anchoring) or FLO1 (for amino terminalanchoring) from S. cerevisiae.

According to yet another embodiment, the fusion protein can comprise afirst and second polypeptide, wherein the first polypeptide comprises aheterologous saccharolytic enzyme and the second polypeptide comprises acellulose binding module (CBM or SBM). In some embodiments, the CBM isfrom, for example, T. reesei Cbh1 or Cbh2 or from C. lucknowense Cbh2b.In some particular embodiments, the CBM is fused to a endoglucanase,glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylanesterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase.

In certain embodiments, the polypeptide of the present inventionencompasses a fusion protein comprising a first polypeptide and a secondpolypeptide, wherein the first polypeptide is an endoglucanase,glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylanesterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase. and the second polypeptideis selected from a polypeptide encoded by a domain or fragment of asaccharolytic enzyme disclosed herein. In certain embodiments, thepolypeptides of the present invention encompasses a fusion proteincomprising a first saccharolytic enzyme polypeptide, where the firstpolypeptide is a domain, derivative or fragment of any saccharolyticenzyme polypeptide disclosed herein, and a second polypeptide, where thesecond polypeptide is a T. emersonii Cbh1, H. grisea Cbh1, or T.aurantiacusi Cbh1, T. emersonii Cbh2, T. reesei Cbh1 or T. reesei Cbh2,C. lucknowense Cbh2b, or domain, fragment, variant, or derivativethereof. In additional embodiments, the first polypeptide is eitherN-terminal or C-terminal to the second polypeptide. In certain otherembodiments, the first polypeptide and/or the second polypeptide areencoded by codon-optimized polynucleotides, for example, polynucleotidescodon-optimized for S. cerevisiae or Kluveromyces.

In certain other embodiments, the first polypeptide and the secondpolypeptide are fused via a linker sequence. The linker sequence can, insome embodiments, be encoded by a codon-optimized polynucelotide.(Codon-optimized polynucleotides are described in more detail below.) Anamino acid sequence corresponding to a codon-optimized linker 1according to the invention is a flexible linker—strep tag—TEVsite—FLAG—flexible linker fusion and corresponds to GGGGSGGGGS AWHPQFGGENLYFQG DYKDDDK GGGGSGGGGS

An exemplary DNA sequence is as follows:

(SEQ ID NO: 41) GGAGGAGGTGGTTCAGGAGGTGGTGGGTCTGCTTGGCATCCACAATTTGGAGGAGGCGGTGGTGAAAATCTGTATTTCCAGGGAGGCGGAGGTGATTACAAGGATGACGACAAAGGAGGTGGTGGATCAGGAGGTGGTGGCTCC

An amino acid sequence corresponding to optimized linker 2 is a flexiblelinker—strep tag—linker—TEV site—flexible linker and corresponds toGGGGSGGGGS WSHPQFEK GG ENLYFQG GGGGSGGGGS. The DNA sequence is asfollows:

ggtggcggtggatctggaggaggcggttcttggtctcacccacaatttgaaaagggtggagaaaacttgtactttcaaggcggtggtggaggttctggcg gaggtggctccggctca.Co-Cultures

In another aspect, the present invention is directed to co-culturescomprising at least two yeast host cells wherein the at least two yeasthost cells each comprise an isolated polynucleotide encoding asaccharolytic enzyme. As used herein, “co-culture” refers to growing twodifferent strains or species of host cells together in the same vessel.In some embodiments of the invention, at least one host cell of theco-culture comprises a heterologous polynucleotide comprising a nucleicacid which encodes an endoglucanase, glucosidase, cellobiohydrolase,xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase,galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase,mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, alpha-glucosidase, pullulanase,isopullulanase, galactosidase, arabinase, arabinoxylanase,arabinosidase, arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase at least one host cell of theco-culture comprises a heterologous polynucleotide comprising a nucleicacid which encodes a different endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,alpha-glucosidase, beta-glucosidase, pullulanase, isopullulanase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and xylose transaldolase and at least one host cellcomprises a heterologous polynucleotide comprising a nucleic acid whichencodes a still different endoglucanase, glucosidase, cellobiohydrolase,xylanase, glucanase, xylosidase, xylan esterase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, alpha-glucosidase, beta-glucosidase,pullulanase, isopullulanase, galactosidase, arabinase, arabinoxylanase,arabinosidase, arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase.

The co-culture can comprise two or more strains of yeast host cells andthe heterologous saccharolytic enzymes can be expressed in anycombination in the two or more strains of host cells. For example,according to the present invention, the co-culture can comprise twostrains: one strain of host cells that expresses an endoglucanase and asecond strain of host cells that expresses a β-glucosidase, acellobiohydrolase and a second cellobiohydrolase. Similarly, theco-culture can comprise one strain of host cells that expresses twosaccharolytic enzymes, for example an endoglucanase and abeta-glucosidase and a second strain of host cells that expresses one ormore saccharolytic enzymes, for example one or more endoglucanase,glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylanesterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase. The co-culture can, inaddition to the at least two host cells comprising heterologoussaccharolytic enzymes, also include other host cells which do notcomprise heterologous saccharolytic enzymes. The co-culture can compriseone strain expressing an endoglucanase, glucosidase, cellobiohydrolase,xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase,galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase,mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, and arabinofuranosidase, arabinose isomerase,ribulose-5-phosphate 4-epimerase, xylose isomerase, xylulokinase, xylosereductase, xylose dehydrogenase, xylitol dehydrogenase, xylonatedehydratase, xylose transketolase, and/or xylose transaldolase; and asecond host cell expressing an endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase.

The various host cell strains in the co-culture can be present in equalnumbers, or one strain or species of host cell can significantlyoutnumber another second strain or species of host cells. For example,in a co-culture comprising two strains or species of host cells theratio of one host cell to another can be about 1:1, 1:2, 1:3, 1:4, 1:5,1:10, 1:100, 1:500 or 1:1000. Similarly, in a co-culture comprisingthree or more strains or species of host cells, the strains or speciesof host cells may be present in equal or unequal numbers.

Biomass feedstocks contain varying proportions of starch,lignocellulose, and pentose sugars. Therefore, in one aspect, yeaststrains express different saccharolytic enzymes at different levels. Inone embodiment, the one or more amylolytic enzymes are expressed athigher levels in yeast strain(s) as compared to one or morelignocellulases and/or the one or more pentose sugar utilizing enzymes.In another embodiment, the one or more lignocellulases are expressed athigher levels in yeast strain(s) as compared to one or more amylolyticenzymes and/or the one or more pentose sugar utilizing enzymes. In yetanother embodiment, the one or more pentose sugar utilizing enzymes areexpressed at higher levels in yeast strain(s) as compared to one or morelignocellulases and/or the one or more amylolytic enzymes. In stillanother embodiment, the one or more amylolytic enzymes, one or morecellulases, and one or more pentose sugar utilizing enzymes are allexpressed at approximately equal levels in the yeast strain(s). In someembodiments of the present invention, the ratio of expression ofamylolytic enzymes to cellulolytic enzymes in the yeast strain(s) isabout 1:5, about 1:2, about 1:1, about 2:1, or about 5:1. In someembodiments of the present invention, the relative expression levels ofthe amylolytic enzymes and cellulolytic enzymes can be determined usingchromatographic techniques, such as HPLC, ion-exchange chromatography,size exclusion chromatography, or by 2D gel electrophoresis,immunoblotting, mass spectrometry, MALDI_TOF, or functional assays.

The co-cultures of the present invention can include tetheredsaccharolytic enzymes, secreted saccharolytic enzymes or both tetheredand secreted saccharolytic enzymes. For example, in some embodiments ofthe invention, the co-culture comprises at least one yeast host cellcomprising a polynucleotide encoding a secreted heterologoussaccharolytic enzymes. In another embodiment, the co-culture comprisesat least one yeast host cell comprising a polynucleotide encoding atethered heterologous saccharolytic enzymes. In one embodiment, all ofthe heterologous saccharolytic enzymes in the co-culture are secreted,and in another embodiment, all of the heterologous saccharolytic enzymesin the co-culture are tethered. In addition, other saccharolyticenzymes, such as externally added saccharolytic enzymes may be presentin the co-culture.

Polynucleotides Encoding Heterologous Saccharolytic Enzymes

In another aspect, the present invention includes isolatedpolynucleotides encoding saccharolytic enzymes of the present invention.Thus, the polynucleotides of the invention can encode endoglucanases,exoglucanases, amylases, or pentose sugar utilizing enzymes. Thepolynucleotides can encode an endoglucanase, glucosidase,cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase,arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase.

The present invention also encompasses an isolated polynucleotidecomprising a nucleic acid that is at least about 70%, 75%, or 80%identical, at least about 90% to about 95% identical, or at least about96%, 97%, 98%, 99% or 100% identical to a nucleic acid encoding anendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, and arabinofuranosidase, arabinose isomerase,ribulose-5-phosphate 4-epimerase, xylose isomerase, xylulokinase, xylosereductase, xylose dehydrogenase, xylitol dehydrogenase, xylonatedehydratase, xylose transketolase, and/or xylose transaldolase disclosedherein.

The present invention also encompasses variants of the saccharolyticenzymes genes, as described above. Variants may contain alterations inthe coding regions, non-coding regions, or both. Examples arepolynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In certain embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. In further embodiments, endoglucanase,glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylanesterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase,cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase,endoxylanase, glucuronidase, acetylxylanesterase,arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin,pectinase, feruoyl esterase, alpha-amylase, beta-amylase, glucoamylase,pullulanase, isopullulanase, alpha-glucosidase, beta-glucosidase,galactosidase, arabinase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinoxylanase, arabinosidase,arabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and xylose transaldolase polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host. Codon-optimized polynucleotides of the presentinvention are discussed further below.

The present invention also encompasses an isolated polynucleotideencoding a fusion protein. In certain embodiments, the nucleic acidencoding a fusion protein comprises a first polynucleotide encoding fora endoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, and arabinofuranosidase, arabinose isomerase,ribulose-5-phosphate 4-epimerase, xylose isomerase, xylulokinase, xylosereductase, xylose dehydrogenase, xylitol dehydrogenase, xylonatedehydratase, xylose transketolase, and xylose transaldolase as disclosedherein and a CBD (as described above).

In further embodiments, the first and second polynucleotides are in thesame orientation, or the second polynucleotide is in the reverseorientation of the first polynucleotide. In additional embodiments, thefirst polynucleotide encodes a polypeptide that is either N-terminal orC-terminal to the polypeptide encoded by the second polynucleotide. Incertain other embodiments, the first polynucleotide and/or the secondpolynucleotide are encoded by codon-optimized polynucleotides, forexample, polynucleotides codon-optimized for S. cerevisiae,Kluyveromyces or for both S. cerevisiae and Kluyveromyces.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to any of SEQ ID NOs: 1-218, or any of SEQ ID NOs:437-441, using information from the sequences disclosed herein or theclones deposited with the ATCC or otherwise publically available. Forexample, allelic variants and/or species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source for allelicvariants and/or the desired homologue.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the particularpolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of any of SEQ ID NOs: 1-218, or any of SEQ IDNOs: 437-441, or any fragment or domain specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence or polypeptide of the presentinvention can be determined conventionally using known computerprograms. A method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Comp. App. Biosci. (1990) 6:237-245.) In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

Some embodiments of the invention encompass a nucleic acid moleculecomprising at least 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, or 800 consecutive nucleotides or more of anyof SEQ ID NOs: 1-218, or any of SEQ ID NOs: 437-441, or domains,fragments, variants, or derivatives thereof.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double stranded or single-stranded, and if singlestranded can be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide can be identical tothe coding sequence encoding SEQ ID NO: 219-436, or SEQ ID NO: 442-446,or may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the samemature polypeptide as the nucleic acid sequences of any one of SEQ IDNOs: 1-218, or any one of SEQ ID NOs: 437-441.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid fragment which encodes at least10, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 95, or at least 100 or morecontiguous amino acids of SEQ ID NOs: 219-436, or SEQ ID NO: 442-446.

The polynucleotide encoding for the mature polypeptide of SEQ ID NOs:219-436, or SEQ ID NO: 442-446 may include: only the coding sequence forthe mature polypeptide; the coding sequence of any domain of the maturepolypeptide; and the coding sequence for the mature polypeptide (ordomain-encoding sequence) together with non coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only sequences encoding for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequences.

In further aspects of the invention, nucleic acid molecules havingsequences at least about 90%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequences disclosed herein, encode a polypeptide having anendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, arabinose isomerase, ribulose-5-phosphate 4-epimerase,xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase,xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, andxylose transaldolase. functional activity.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large portion of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of any of SEQ ID NOs:1-218, or any of SEQ ID NOs: 437-441, or fragments thereof, will encodepolypeptides having functional activity. In fact, since degeneratevariants of any of these nucleotide sequences all encode the samepolypeptide, in many instances, this will be clear to the skilledartisan even without performing the above described comparison assay. Itwill be further recognized in the art that, for such nucleic acidmolecules that are not degenerate variants, a reasonable number willalso encode a polypeptide having functional activity.

The polynucleotides of the present invention also comprise nucleic acidsencoding an endoglucanase, glucosidase, cellobiohydrolase, xylanase,glucanase, xylosidase, xylan esterase, arabinofuranosidase,galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase,mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, arabinose isomerase, ribulose-5-phosphate 4-epimerase,xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase,xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, andxylose transaldolase, or domain, fragment, variant, or derivativethereof, fused to a polynucleotide encoding a marker sequence whichallows for detection of the polynucleotide of the present invention. Inone embodiment of the invention, expression of the marker is independentfrom expression of the saccharolytic enzyme. The marker sequence may bea yeast selectable marker selected from the group consisting of URA3,HIS3, LEU2, TRP1, LYS2, ADE2 or any other suitable selectable markerknown in the art. Casey, G. P. et al., “A convenient dominant selectionmarker for gene transfer in industrial strains of Saccharomyces yeast:SMR1 encoded resistance to the herbicide sulfometuron methyl,” J. Inst.Brew. 94:93-97 (1988).

Codon Optimized Polynucleotides

According to one embodiment of the invention, the polynucleotidesencoding heterologous saccharolytic enzymes can be codon-optimized. Asused herein the term “codon-optimized coding region” means a nucleicacid coding region that has been adapted for expression in the cells ofa given organism by replacing at least one, or more than one, or asignificant number, of codons with one or more codons that are morefrequently used in the genes of that organism.

In general, highly expressed genes in an organism are biased towardscodons that are recognized by the most abundant tRNA species in thatorganism. One measure of this bias is the “codon adaptation index” or“CAI,” which measures the extent to which the codons used to encode eachamino acid in a particular gene are those which occur most frequently ina reference set of highly expressed genes from an organism.

The CAI of codon optimized sequences of the present inventioncorresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, orabout 1.0. A codon optimized sequence may be further modified forexpression in a particular organism, depending on that organism'sbiological constraints. For example, large runs of “As” or “Ts” (e.g.,runs greater than 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can beremoved from the sequences if these are known to effect transcriptionnegatively. Furthermore, specific restriction enzyme sites may beremoved for molecular cloning purposes. Examples of such restrictionenzyme sites include PacI, AscI, BamHI, BglII, EcoRI and XhoI.Additionally, the DNA sequence can be checked for direct repeats,inverted repeats and mirror repeats with lengths of ten bases or longer,which can be modified manually by replacing codons with “second best”codons, i.e., codons that occur at the second highest frequency withinthe particular organism for which the sequence is being optimized.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W)C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro(P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg(R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACTThr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGCSer (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACGThr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D)GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V)GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E)GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage Tables are readilyavailable, for example, athttp://phenotype.biosci.umbc.edu/codon/sgd/index.php (visited May 7,2008) or at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), andthese tables can be adapted in a number of ways. See Nakamura, Y., etal., “Codon usage tabulated from the international DNA sequencedatabases: status for the year 2000,” Nucl. Acids Res. 28:292 (2000).Codon usage tables for yeast, calculated from GenBank Release 128.0 [15Feb. 2002], are reproduced below as Table 2. This Table uses mRNAnomenclature, and so instead of thymine (T) which is found in DNA, thetables use uracil (U) which is found in RNA. The Table has been adaptedso that frequencies are calculated for each amino acid, rather than forall 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Genes Frequencyper Amino Acid Codon Number hundred Phe UUU 170666 26.1 Phe UUC 12051018.4 Total Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3Leu CUC 35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Total Ile AUU196893 30.1 Ile AUC 112176 17.2 Ile AUA 116254 17.8 Total Met AUG 13680520.9 Total Val GUU 144243 22.1 Val GUC 76947 11.8 Val GUA 76927 11.8 ValGUG 70337 10.8 Total Ser UCU 153557 23.5 Ser UCC 92923 14.2 Ser UCA122028 18.7 Ser UCG 55951 8.6 Ser AGU 92466 14.2 Ser AGC 63726 9.8 TotalPro CCU 88263 13.5 Pro CCC 44309 6.8 Pro CCA 119641 18.3 Pro CCG 345975.3 Total Thr ACU 132522 20.3 Thr ACC 83207 12.7 Thr ACA 116084 17.8 ThrACG 52045 8.0 Total Ala GCU 138358 21.2 Ala GCC 82357 12.6 Ala GCA105910 16.2 Ala GCG 40358 6.2 Total Tyr UAU 122728 18.8 Tyr UAC 9659614.8 Total His CAU 89007 13.6 His CAC 50785 7.8 Total Gln CAA 17825127.3 Gln CAG 79121 12.1 Total Asn AAU 233124 35.7 Asn AAC 162199 24.8Total Lys AAA 273618 41.9 Lys AAG 201361 30.8 Total Asp GAU 245641 37.6Asp GAC 132048 20.2 Total Glu GAA 297944 45.6 Glu GAG 125717 19.2 TotalCys UGU 52903 8.1 Cys UGC 31095 4.8 Total Trp UGG 67789 10.4 Total ArgCGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 19562 3.0 Arg CGG 11351 1.7 ArgAGA 139081 21.3 Arg AGG 60289 9.2 Total Gly GGU 156109 23.9 Gly GGC63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 Total Stop UAA 6913 1.1Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar Tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.Codon-optimized coding regions can be designed by various differentmethods.

In one method, a codon usage Table is used to find the single mostfrequent codon used for any given amino acid, and that codon is usedeach time that particular amino acid appears in the polypeptidesequence. For example, referring to Table 2 above, for leucine, the mostfrequent codon is UUG, which is used 27.2% of the time. Thus all theleucine residues in a given amino acid sequence would be assigned thecodon UUG.

In another method, the actual frequencies of the codons are distributedrandomly throughout the coding sequence. Thus, using this method foroptimization, if a hypothetical polypeptide sequence had 100 leucineresidues, referring to Table 2 for frequency of usage in the S.cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11,or 11% of the leucine codons would be CUG, about 12, or 12% of theleucine codons would be CUU, about 13, or 13% of the leucine codonswould be CUA, about 26, or 26% of the leucine codons would be UUA, andabout 27, or 27% of the leucine codons would be UUG.

These frequencies would be distributed randomly throughout the leucinecodons in the coding region encoding the hypothetical polypeptide. Aswill be understood by those of ordinary skill in the art, thedistribution of codons in the sequence can vary significantly using thismethod; however, the sequence always encodes the same polypeptide.

When using the methods above, the term “about” is used precisely toaccount for fractional percentages of codon frequencies for a givenamino acid. As used herein, “about” is defined as one amino acid more orone amino acid less than the value given. The whole number value ofamino acids is rounded up if the fractional frequency of usage is 0.50or greater, and is rounded down if the fractional frequency of use is0.49 or less. Using again the example of the frequency of usage ofleucine in human genes for a hypothetical polypeptide having 62 leucineresidues, the fractional frequency of codon usage would be calculated bymultiplying 62 by the frequencies for the various codons. Thus, 7.28percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUAcodons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e.,7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or“about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percentof 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons,and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e.,24, 25, or 26 CUG codons.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTl Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG—Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences, e.g., the “backtranslation” function athttp://www.entelechon.com/2008/10/backtranslation-tool/ (visited May 30,2010). Constructing a rudimentary algorithm to assign codons based on agiven frequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

A number of options are available for synthesizing codon optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art. In one approach, a series ofcomplementary oligonucleotide pairs of 80-90 nucleotides each in lengthand spanning the length of the desired sequence is synthesized bystandard methods. These oligonucleotide pairs are synthesized such thatupon annealing, they form double stranded fragments of 80-90 base pairs,containing cohesive ends, e.g., each oligonucleotide in the pair issynthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond theregion that is complementary to the other oligonucleotide in the pair.The single-stranded ends of each pair of oligonucleotides is designed toanneal with the single-stranded end of another pair of oligonucleotides.The oligonucleotide pairs are allowed to anneal, and approximately fiveto six of these double-stranded fragments are then allowed to annealtogether via the cohesive single stranded ends, and then they ligatedtogether and cloned into a standard bacterial cloning vector, forexample, a TOPO® vector available from Invitrogen Corporation, Carlsbad,Calif. The construct is then sequenced by standard methods. Several ofthese constructs consisting of 5 to 6 fragments of 80 to 90 base pairfragments ligated together, i.e., fragments of about 500 base pairs, areprepared, such that the entire desired sequence is represented in aseries of plasmid constructs. The inserts of these plasmids are then cutwith appropriate restriction enzymes and ligated together to form thefinal construct. The final construct is then cloned into a standardbacterial cloning vector, and sequenced. Additional methods would beimmediately apparent to the skilled artisan. In addition, gene synthesisis readily available commercially.

In certain embodiments, an entire polypeptide sequence, or fragment,variant, or derivative thereof is codon optimized by any of the methodsdescribed herein. Various desired fragments, variants or derivatives aredesigned, and each is then codon-optimized individually. In addition,partially codon-optimized coding regions of the present invention can bedesigned and constructed. For example, the invention includes a nucleicacid fragment of a codon-optimized coding region encoding a polypeptidein which at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the codon positions have been codon-optimized for a given species.That is, they contain a codon that is preferentially used in the genesof a desired species, e.g., a yeast species such as Saccharomycescerevisiae or Kluveromyces, in place of a codon that is normally used inthe native nucleic acid sequence.

In additional embodiments, a full-length polypeptide sequence iscodon-optimized for a given species resulting in a codon-optimizedcoding region encoding the entire polypeptide, and then nucleic acidfragments of the codon-optimized coding region, which encode fragments,variants, and derivatives of the polypeptide are made from the originalcodon-optimized coding region. As would be well understood by those ofordinary skill in the art, if codons have been randomly assigned to thefull-length coding region based on their frequency of use in a givenspecies, nucleic acid fragments encoding fragments, variants, andderivatives would not necessarily be fully codon optimized for the givenspecies. However, such sequences are still much closer to the codonusage of the desired species than the native codon usage. The advantageof this approach is that synthesizing codon-optimized nucleic acidfragments encoding each fragment, variant, and derivative of a givenpolypeptide, although routine, would be time consuming and would resultin significant expense.

The codon-optimized coding regions can be, for example, versionsencoding an endoglucanase, glucosidase, cellobiohydrolase, xylanase,glucanase, xylosidase, xylan esterase, arabinofuranosidase,galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase,mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, feruoyl esterase, alpha-amylase,beta-amylase, glucoamylase, pullulanase, isopullulanase,alpha-glucosidase, beta-glucosidase, galactosidase, arabinase,arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase,arabinosidase, and arabinofuranosidase, arabinose isomerase,ribulose-5-phosphate 4-epimerase, xylose isomerase, xylulokinase, xylosereductase, xylose dehydrogenase, xylitol dehydrogenase, xylonatedehydratase, xylose transketolase, and/or xylose transaldolase asdisclosed herein, or domains, fragments, variants, or derivativesthereof.

Codon optimization is carried out for a particular species by methodsdescribed herein, for example, in certain embodiments codon-optimizedcoding regions encoding polypeptides disclosed in the presentapplication or domains, fragments, variants, or derivatives thereof areoptimized according to yeast codon usage, e.g., Saccharomycescerevisiae, Kluyveromyces lactis and/or Kluyveromyces marxianus. Alsoprovided are polynucleotides, vectors, and other expression constructscomprising codon-optimized coding regions encoding polypeptidesdisclosed herein, or domains, fragments, variants, or derivativesthereof, and various methods of using such polynucleotides, vectors andother expression constructs.

In certain embodiments described herein, a codon-optimized coding regionencoding any of SEQ ID NOs: 219-436, or any of SEQ ID NOs: 442-446, ordomain, fragment, variant, or derivative thereof, is optimized accordingto codon usage in yeast (e.g. Saccharomyces cerevisiae, Kluyveromyceslactis or Kluyveromyces marxianus). In some embodiments, the sequencesare codon-optimized specifically for expression in Saccharomycescerevisiae. Alternatively, a codon-optimized coding region encoding anyof SEQ ID NOs: 219-436, or any of SEQ ID NOs: 442-446 may be optimizedaccording to codon usage in any plant, animal, or microbial species.

Vectors and Methods of Using Vectors in Host Cells

In another aspect, the present invention relates to vectors whichinclude polynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention can be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; and yeast plasmids. However, any other vector may beused as long as it is replicable and viable in the host.

The appropriate DNA sequence can be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively associated withan appropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. Representative examples of such promoters are as follows:

Gene Organism Systematic name Reason for use/benefits PGK1 S. cerevisiaeYCR012W Strong constitutive promoter ENO1 S. cerevisiae YGR254W Strongconstitutive promoter TDH3 S. cerevisiae YGR192C Strong constitutivepromoter TDH2 S. cerevisiae YJR009C Strong constitutive promoter TDH1 S.cerevisiae YJL052W Strong constitutive promoter ENO2 S. cerevisiaeYHR174W Strong constitutive promoter GPM1 S. cerevisiae YKL152C Strongconstitutive promoter TPI1 S. cerevisiae YDR050C Strong constitutivepromoter

Additionally, promoter sequences from stress and starvation responsegenes are useful in the present invention. In some embodiments, promoterregions from the S. cerevisiae genes GAC1, GET3, GLC7, GSH1, GSH2, HSF1,HSP12, LCB5, LRE1, LSP1, NBP2, PDC1, PIL1, PIM1, SGT2, SLG1, WHI2, WSC2,WSC3, WSC4, YAP1, YDC1, HSP104, HSP26, ENA1, MSN2, MSN4, SIP2, SIP4,SIP5, DPL1, IRS4, KOG1, PEP4, HAP4, PRB1, TAX4, ZPR1, ATG1, ATG2, ATG10,ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, and ATG19 may beused. Any suitable promoter to drive gene expression in the host cellsof the invention may be used. Additionally the E. coli, lac or trp, andother promoters known to control expression of genes in prokaryotic orlower eukaryotic cells can be used.

In addition, the expression vectors may contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells such as URA3, HIS3, LEU2, TRP1, LYS2 or ADE2, dihydrofolatereductase, neomycin (G418) resistance or zeocin resistance foreukaryotic cell culture, or tetracycline or ampicillin resistance in E.coli.

The expression vector may also contain a ribosome binding site fortranslation initiation and/or a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression, or mayinclude additional regulatory regions.

The vector containing the appropriate DNA sequence as disclosed herein,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express theprotein.

Thus, in certain aspects, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a hostcell as described elsewhere in the application. The host cell can be,for example, a lower eukaryotic cell, such as a yeast cell, e.g.,Saccharomyces cerevisiae or Kluyveromyces, or the host cell can be aprokaryotic cell, such as a bacterial cell.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;thermophilic or mesophlic bacteria; fungal cells, such as yeast; andplant cells, etc. The selection of an appropriate host is deemed to bewithin the scope of those skilled in the art from the teachings herein.

Appropriate fungal hosts include yeast. In certain aspects of theinvention the yeast is selected from the group consisting ofSaccharomyces cerevisiae, Kluyveromyces lactis, Schizzosaccharomycespombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowialipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis,Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus,Schwanniomyces occidentalis, Issatchenkia orientalis, Kluyveromycesmarxianus, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces,Mortierella, Mucor, Phycomces, Pythium, Rhodosporidium, Rhodotorula,Trichosporon and Yarrowia.

Methods of Using Host Cells to Produce Ethanol or Other FermentationProducts

In another aspect, the present invention is directed to the use of hostcells and co-cultures to produce ethanol or other products from abiomass feedstock comprising starch, lignocellulosic matter, hexose andpentose sugars. Such methods can be accomplished, for example, bycontacting a biomass feedstock with a host cell or a co-culture of thepresent invention. Fermentation products include, but are not limited toproducts such as butanol, acetate, amino acids, and vitamins.

Numerous biomass feedstocks can be used in accordance with the presentinvention. Substrates for saccharolytic enzyme activity assays can bedivided into two categories, soluble and insoluble, based on theirsolubility in water. Soluble substrates include alpha-dextrins,cellodextrins or derivatives, carboxymethyl cellulose (CMC), orhydroxyethyl cellulose (HEC). Insoluble substrates include insolublestarch, crystalline cellulose, microcrystalline cellulose (Avicel),amorphous cellulose, such as phosphoric acid swollen cellulose (PASC),dyed or fluorescent cellulose, and lignocellulosic biomass. Thesesubstrates are generally highly ordered cellulosic material and thusonly sparingly soluble.

It will be appreciated that suitable lignocellulosic material may be anyfeedstock that contains soluble and/or insoluble cellulose, where theinsoluble cellulose may be in a crystalline or non-crystalline form. Invarious embodiments, the lignocellulosic biomass comprises, for example,wood, corn, corn stover, sawdust, bark, leaves, agricultural andforestry residues, grasses such as switchgrass, ruminant digestionproducts, municipal wastes, paper mill effluent, newspaper, cardboard orcombinations thereof.

In some embodiments, the invention is directed to a method forhydrolyzing a biomass feedstock, for example a biomass feedstock asdescribed above, by contacting the biomass feedstock with a host cell ofthe invention. In some embodiments, the invention is directed to amethod for hydrolyzing a biomass feedstock, for example a biomassfeedstock as described above, by contacting the feedstock with aco-culture comprising yeast cells expressing heterologous saccharolyticenzymes.

In some embodiments of the present invention, the necessity of addingexternal saccharolytic enzymes to the fermentation medium is reducedbecause cells of the invention express polypeptides of the invention.

In some embodiments, the invention is directed to a method forfermenting a biomass feedstock. Such methods can be accomplished, forexample, by culturing a host cell or co-culture in a medium thatcontains insoluble biomass feedstock to allow saccharification andfermentation of the biomass feedstock.

In addition to the enzymes of the present invention, in someembodiments, host cells of the present invention can have furthergenetic modifications to make them more suitable for fermenting biomassfeedstock to ethanol. For example, host cells of the present inventionmay express xylose isomerase and/or arabinose isomerase inorder to moreefficiently use pentose sugars for fermentation. In some embodiments,the xylose isomerase is from a Pyromyces species. In addition to axylose isomerase, host cells of the invention, in some embodiments, canover-express genes related to the pentose phosphate pathway. These genesinclude, but are not limited to transkelolase and transaldolase genes.Components of the pentose phosphate pathway are known to those skilledin the art and are useful in aiding assimilation of carbons derived frompentose sugars into fermentation processes. (See, e.g. WO 03/062430, WO06/009434, and US 2006/0234364). In some embodiments, a host cell isable to use xylose and other pentose sugars such as arabinose byincorporating the carbons from pentose sugars into fermentative pathwaysvia the pentose phosphate pathway. The xylose-utilizing host cellheterologously expresses xylose isomerase, e.g. Pyromyces sp. E2 XylA,overexpresses xylulokinase, ribulose 5-phosphate isomerase, ribulose5-phophate epimerase, transketolase and transaldolase, and does notexpress an aldose reductase such as the GRE3 gene (encoding an aldosereductase).

The production of ethanol can, according to the present invention, beperformed at temperatures of at least about 25° C., about 28° C., about30° C., about 31° C., about 32° C., about 33° C., about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., about40° C., about 41° C., about 42° C., or about 50° C. In some embodimentsof the present invention, the thermotolerant host cell can produceethanol from cellulose at temperatures above about 30° C., about 31° C.,about 32° C., about 33° C., about 34° C., about 35° C., about 36° C.,about 37° C., about 38° C., about 39° C., about 40° C., about 41° C.,about 42° C., or about 50° C. In some embodiments of the presentinvention, the thermotolterant host cell can produce ethanol fromcellulose at temperatures from about 30° C. to 60° C., about 30° C. to55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C. to55° C. or about 40° C. to 50° C.

In some embodiments, methods of producing ethanol can comprisecontacting a biomass feedstock with a host cell or co-culture of theinvention and additionally contacting the biomass feedstock withexternally produced saccharolytic enzymes. Exemplary externally producedsaccharolytic enzymes are commercially available and are known to thoseof skill in the art and are further exemplified below.

Therefore, the invention is also directed to methods of reducing theamount of externally produced saccharolytic enzymes required to producea given amount of ethanol from the biomass feedstock comprisingcontacting the saccharolytic enzyme with externally producedsaccharolytic enzymes and with a host cell or co-culture of theinvention. In some embodiments, the same amount of ethanol productioncan be achieved using at least about 5%, 10%, 15%, 20%, 25%, 30%, or 50%fewer externally produced saccharolytic enzymes.

In some embodiments, the methods comprise producing ethanol at aparticular rate. For example, in some embodiments, ethanol is producedat a rate of at least about 0.1 mg per hour per liter, at least about0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, atleast about 0.75 mg per hour per liter, at least about 1.0 mg per hourper liter, at least about 2.0 mg per hour per liter, at least about 5.0mg per hour per liter, at least about 10 mg per hour per liter, at leastabout 15 mg per hour per liter, at least about 20.0 mg per hour perliter, at least about 25 mg per hour per liter, at least about 30 mg perhour per liter, at least about 50 mg per hour per liter, at least about100 mg per hour per liter, at least about 200 mg per hour per liter, orat least about 500 mg per hour per liter.

In some embodiments, the host cells of the present invention can produceethanol at a rate of at least about 0.1 mg per hour per liter, at leastabout 0.25 mg per hour per liter, at least about 0.5 mg per hour perliter, at least about 0.75 mg per hour per liter, at least about 1.0 mgper hour per liter, at least about 2.0 mg per hour per liter, at leastabout 5.0 mg per hour per liter, at least about 10 mg per hour perliter, at least about 15 mg per hour per liter, at least about 20.0 mgper hour per liter, at least about 25 mg per hour per liter, at leastabout 30 mg per hour per liter, at least about 50 mg per hour per liter,at least about 100 mg per hour per liter, at least about 200 mg per hourper liter, or at least about 500 mg per hour per liter more than acontrol strain (lacking heterologous biomass feedstock hydrolyzingenzymes) and grown under the same conditions. In some embodiments, theethanol can be produced in the absence of any externally addedsaccharolytic enzymes.

Ethanol production can be measured using any method known in the art.For example, the quantity of ethanol in fermentation samples can beassessed using HPLC analysis. Many ethanol assay kits are commerciallyavailable that use, for example, alcohol oxidase enzyme based assays.Methods of determining ethanol production are within the scope of thoseskilled in the art from the teachings herein.

Synergistic Activity of Sacchcarolytic Enzymes

In some embodiments, the expression of two or more enzymes of thepresent invention results in synergistic enzymatic activity with respectto substrate digestion. For example, the presence of two distinctparalogs or orthologs containing the same enzymatic activity cansignificantly enhance the digestion of a substrate compared to acomparable amount of either enzyme by itself. Alternatively,synergistically acting enzymes do not need to have exactly identicalchemical activity, but can still operate to liberate sugars in acapacity greater than either is capable of individually. Without wishingto be bound by a particular theory, it is thought that although thecatalytic activity of the enzymes can be the same, the differentcharacteristics of the enzymes with respect to the regions surroundingthe chemical substrate as well as other differing properties of theenzymes aid in digesting the varied biomass feedstock components. Insome embodiments, enzymatic synergy allows biomass feedstock digestionand fermentation to take place using reduced amounts of externalsaccharolytic enzymes. In some embodiments, the two or more enzymesacting synergistically are endoglucanases, glucosidases,cellobiohydrolases, xylanases, glucanases, xylosidases, xylan esterases,arabinofuranosidases, galactosidases, cellobiose phosphorylases,cellodextrin phosphorylases, mannanases, mannosidases, xyloglucanases,endoxylanases, glucuronidases, acetylxylanesterases,arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins,pectinases, feruoyl esterases, alpha-amylase, beta-amylase,glucoamylase, pullulanase, isopullulanase, alpha-glucosidase,beta-glucosidase, galactosidase, arabinase, arabinoxylanase,arabinosidase, arabinofuranosidase, arabinoxylanase, arabinosidase,arabinose isomerase, ribulose-5-phosphate 4-epimerase, xylose isomerase,xylulokinase, xylose reductase, xylose dehydrogenase, xylitoldehydrogenase, xylonate dehydratase, xylose transketolase, and/or xylosetransaldolase as disclosed herein. In some embodiments, the two or moreenzymes acting synergistically do not have the same enzymatic activity.In other embodiments, the two or more enzymes acting synergisticallyhave the same enzyme activity. In some embodiments, the enzyme pairsacting synergistically are (Streptomyces avermitilisendo-1,4-beta-glucanase celA2 (Accession No. NP_823030.1) andStreptomyces avermitilis endo-1,4-beta-glucanase celA5 (Accession No.NP_828072.1)); (Streptomyces avermitilis endo-1,4-beta-glucanase celA2(Accession No. NP_823030.1) and Bacillus subtilisendo-1,4-beta-glucanase (Accession No CAB13696.2)); (Streptomycesavermitilis endo-1,4-beta-glucanase celA3 (Accession No. NP_823032.1)and Streptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_826394.1)); (Streptomyces avermitilis endo-1,4-beta-glucanase celA4(Accession No. NP_823744.1) and Streptomyces avermitilis xylanase(Accession No. NP_827548.1)); (Bacillus subtilis endo-1,4-beta-glucanase(Accession No CAB13696.2) and Streptomyces avermitilisendo-1,4-beta-glucanase (Accession No. NP_826394.1)); (Streptomycesavermitilis endo-1,4-beta-glucanase celA4 (Accession No. NP_823744.1)and Bacillus subtilis endo-1,4-beta-glucanase (Accession NoCAB13696.2)); (Streptomyces avermitilis endo-1,4-beta-glucanase celA5(Accession No. NP_828072.1) and Streptomyces avermitilisendo-1,4-beta-glucanase celA4 (Accession No. NP_823744.1));(Streptomyces avermitilis endo-1,4-beta-glucanase celA5 (Accession No.NP_828072.1) and Clostridium phytofermentans xylanase (Accession No.YP_001557750.1)); (Saccharophagus degradans 2-40 mannanase (AccessionNo. YP_525985.1) and Streptomyces avermitilis endo-1,4-beta-glucanase(Accession No. NP_826394.1)); (Streptomyces avermitilis xylanase(Accession No. NP_827548.1) and Saccharophagus degradans 2-40 mannanase(Accession No. YP_525985.1)); (Clostridium phytofermentans xylanase(Accession No. YP_001557750.1) and Streptomyces avermitilis xylanase(Accession No. NP_827548.1)); (Clostridium phytofermentans xylanase(Accession No. YP_001557750.1) and Streptomyces avermitilis xylanase(Accession No. NP_827548.1)); (Streptomyces avermitilisendo-1,4-beta-glucanase celA5 (Accession No. NP_828072.1) andStreptomyces avermitilis xylanase (Accession No. NP_827548.1));(Streptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_823744.1) and Saccharophagus degradans 2-40 mannanase (Accession No.YP_525985.1)); (Streptomyces avermitilis endo-1,4-beta-glucanase celA2(Accession No. NP_823030.1) and Saccharophagus degradans 2-40 mannanase(Accession No. YP_525985.1)); (Streptomyces avermitilisendo-1,4-beta-glucanase (Accession No. NP_823744.1) and Streptomycesavermitilis endo-1,4-beta-glucanase celA3 (Accession No. NP_823032.1));(Streptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_823744.1) and Clostridium phytofermentans xylanase (Accession No.YP_001557750.1)); (Streptomyces avermitilis xylanase (Accession No.NP_827548.1) and Streptomyces avermitilis endo-1,4-beta-glucanase celA3(Accession No. NP_823032.1)); or (Streptomyces avermitilisendo-1,4-beta-glucanase celA4 (Accession No. NP_823744.1) andStreptomyces avermitilis endo-1,4-beta-glucanase (Accession No.NP_826394.1)); (SEQ ID NO: 443 and SEQ ID NO: 444); (SEQ ID NO: 443 andSEQ ID NO: 445); (SEQ ID NO: 445 and SEQ ID NO: 446); (SEQ ID NO: 443and SEQ ID NO: 445); (SEQ ID NO: 442 and SEQ ID NO: 445); (SEQ ID NO:444 and Bacillus subtilis arabinoxylanase (Accession No. CAB13699.1));(SEQ ID NO: 444 and Bacillus subtilis arabinoxylanase (Accession No.CAB13699.1)); (SEQ ID NO: 444 and Bacillus subtilis arabinanendo-1,5-alpha-L-arabinosidase (Accession No. CAB15969.1)); (SEQ ID NO:444 and Bacillus subtilis arabinan-endo 1,5-alpha-L-arabinase (AccessionNo. CAA99586.1)); (SEQ ID NO: 444 and Bacillus subtilis arabinanendo-1,5-alpha-L-arabinosidase (Accession No. AL009126)); (SEQ ID NO:444 and Bacillus subtilis endo-arabinase (Accession No. D85132)); (SEQID NO: 444 and Clostridium phytofermentans arabinogalactanendo-1,4-beta-galactosidase (Accession No. CP000885)); (SEQ ID NO: 444and Bacillus licheniformis arabinan-endo 1,5-alpha-L-arabinase(Accession No. AAU40201.1); (SEQ ID NO: 444 and Bacillus licheniformisarabinan-endo 1,5-alpha-L-arabinase (Accession No. AAU41895.1); (SEQ IDNO: 444 and Bacillus licheniformis arabinogalactanendo-1,4-beta-galactosidase (Accession No. AAU43089.1); (SEQ ID NO: 444and Bacillus licheniformis arabinan endo-1,5-alpha-L-arabinosidase(Accession No. AAU43033.1); (SEQ ID NO: 444 and Bacillus licheniformisarabinan endo-1,4-beta-xylanase (Accession No. AAU39947.1); (SEQ ID NO:444 and Thermoanaerobacterium saccharolyticum arabinogalactanendo-1,4-beta-galactosidase; (SEQ ID NO: 444 and Thermoanaerobacteriumsaccharolyticum alpha-N-arabinofuranosidase); (SEQ ID NO: 444 andStreptomyces avermitilis endo-1,4-beta-xylanase xynD (Accession No.827557.1); (SEQ ID NO: 444 and Bacillus subtilis endo-1,4-beta-xylanasexynA (Accession No. CAB13776.1); (SEQ ID NO: 444 and Clostridiumphytofermentans xylanase (Accession No. YP_001558623.1); (SEQ ID NO: 444and Clostridium phytofermentans xylanase (Accession No. YP_001557750.1);(SEQ ID NO: 444 and Thermobifida fusca endo-1,4-beta-D-xylanase (xyl11)(Accession No. AAV64879.1); (SEQ ID NO: 444 and Clostridium thermocellumxylanase (Accession No. YP_001038519.1); (SEQ ID NO: 444 and Clostridiumstercorarium endo-xylanase (Accession No. CAD48307); (SEQ ID NO: 444 andClostridium stercorarium xynC (CelX—celloxylanase) (Accession No.CAD48314); (SEQ ID NO: 444 and Aspergillus niger alpha-glucosidase(Accession No. BAA23616.1)); (SEQ ID NO: 444 and Thermoanaerobacteriumsaccharolyticum glucoamylase).

In other embodiments, the enzyme triplets acting synergisticallyinclude, but are not limited to (SEQ ID NO: 442, SEQ ID NO: 445 and SEQID NO: 446); (SEQ ID NO: 444, SEQ ID NO: 445 and SEQ ID NO: 446); or(SEQ ID NO: 442, SEQ ID NO: 445 and SEQ ID NO: 446).

In yet other embodiments, the enzyme combinations acting synergisticallyinclude, but are not limited to (SEQ ID NO: 442, SEQ ID NO: 444, SEQ IDNO: 445 and SEQ ID NO: 446); (SEQ ID NO: 443, SEQ ID NO: 444, SEQ ID NO:445 and SEQ ID NO: 446).

In other embodiments, enzymatic synergy may be achieved by expressing 3,4, 5, 6, or 7 or more enzymes with the same catalytic activity. In oneembodiment, two or more enzymes acting synergistically with sameenzymatic activity include, but are not limited to (SEQ ID NO: 444 andSEQ ID NO: 444); (SEQ ID NO: 445 and SEQ ID NO: 445).

Glycerol Reduction

Anaerobic growth conditions require the production of endogenouseelectron acceptors, such as the coenzyme nicotinamide adeninedinucleotide (NAD⁺). In cellular redox reactions, the NAD⁺/NADH coupleplays a vital role as a reservoir and carrier of reducing equivalents.Ansell, R., et al., EMBO J. 16:2179-87 (1997). Cellular glycerolproduction, which generates an NAD⁺, serves as a redox valve to removeexcess reducing power during anaerobic fermentation in yeast. Glycerolproduction is, however, an energetically wasteful process that expendsATP and results in the loss of a reduced three-carbon compound. Ansell,R., et al., EMBO J. 16:2179-87 (1997). To generate glycerol from astarting glucose molecule, glycerol 3-phosphate dehydrogenase (GPD)reduces dihydroxyacetone phosphate to glycerol 3-phosphate and glycerol3-phosphatase (GPP) dephosphorylates glycerol 3-phosphate to glycerol.Despite being energetically wasteful, glycerol production is a necessarymetabolic process for anaerobic growth as deleting GPD activitycompletely inhibits growth under anaeroblic conditions. See Ansell, R.,et al., EMBO J. 16:2179-87 (1997).

GPD is encoded by two isogenes, gpd1 and gpd2. GPD1 encodes the majorisoform in anaerobically growing cells, while GPD2 is required forglycerol production in the absence of oxygen, which stimulates itsexpression. Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001).The first step in the conversion of dihydroxyacetone phosphate toglycerol by GPD is rate controlling. Guo, Z. P., et al., Metab. Eng.13:49-59 (2011). GPP is also encoded by two isogenes, gpp1 and gpp2. Thedeletion of GPP genes arrests growth when shifted to anaerobicconditions, demonstrating that GPP is important for cellular toleranceto osmotic and anaerobic stress. See Pahlman, A-K., et al., J. Biol.Chem. 276:3555-63 (2001).

Because glycerol is a major by-product of anaerobic production ofethanol, many efforts have been made to delete cellular production ofglycerol. However, because of the reducing equivalents produced byglycerol synthesis, deletion of the glycerol synthesis pathway cannot bedone without compensating for this valuable metabolic function. Attemptsto delete glycerol production and engineer alternate electron acceptorshave been made. Liden, G., et al., Appl. Env. Microbiol. 62:3894-96(1996); Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010).Liden and Medina both deleted the gpd1 and gpd2 genes and attempted tobypass glycerol formation using additional carbon sources. Lidenengineered a xylose reductase from Pichia stipitis into an S. cerevisiaegpd1/2 deletion strain. The xylose reductase activity facilitated theanaerobic growth of the glycerol-deleted strain in the presence ofxylose. See Liden, G., et al., Appl. Env. Microbiol. 62:3894-96 (1996).Medina engineered an acetylaldehyde dehydrogenase, mhpF, from E. coliinto an S. cerevisiae gpd1/2 deletion strain to convert acetyl-CoA toacetaldehyde. The acetylaldehyde dehydrogenase activity facilitated theanaerobic growth of the glycerol-deletion strain in the presence ofacetic acid but not in the presence of glucose as the sole source ofcarbon. Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010);see also EP 2277989. Medina noted several issues with themhpF-containing strain that needed to be addressed before implementingindustrially, including significantly reduced growth and productformation rates than yeast comprising GPD1 and GPD2.

Additional attempts to redirect flux from glycerol to ethanol haveincluded the engineering of a non-phosphorylating NADP+-dependentglyceraldehydes-3-phosphate dehydrogenase (GAPN) into yeast, either withor without the simultaneous knockout of GPD1. Bro, C., et al., Metab.Eng. 8:102-111 (2006); U.S. Patent Appl. Pub. No. US2006/0257983; Guo,Z. P., et al., Metab. Eng. 13:49-59 (2011). However, other cellularmechanisms exist to control the production and accumulation of glycerol,including glycerol exporters such as FPS1, that do not require theengineering of alternate NADP+/NADPH coupling or deletion of glycerolsynthesis genes. Tamás, M. J., et al., Mol. Microbiol. 31:1087-1004(1999).

FPS1 is a channel protein located in the plasma membrane that controlsthe accumulation and release of glycerol in yeast osmoregulation. Nullmutants of this strain accumulate large amounts of intracellularglycerol, grow much slower than wild-type, and consume the sugarsubstrate at a slower rate. Minas, M. J., et al., Mol. Microbiol.31:1087-1004 (1999). Despite slower growth under anaerobic conditions,an fps1Δ strain can serve as an alternative to eliminatingNAD⁺-dependant glycerol activity. An fps1Δ strain has reduced glycerolformation yet has a completely functional NAD⁺-dependant glycerolsynthesis pathway. Alternatively, rather than deleting endogenous FPS1,constitutively active mutants of FPS1 or homologs from other organismscan be used to regulate glycerol synthesis while keep the NAD⁺-dependantglycerol activity intact. In embodiments of the invention that modulateFPS1, the recombinant host cells can still synthesize and retainglycerol and achieve improved robustness relative to strains that areunable to make glycerol.

In one embodiment, one or more endogenous glycerol-producing orregulating genes are deleted to create yeast strains with alteredglycerol production. In another embodiment, one or more endogenousglycerol-producing genes are downregulated to create yeast strains withaltered glycerol production. In still another embodiment, one or moreendogenous glycerol-regulating genes are downregulated to create yeaststrains with altered glycerol production. In yet another embodiment, oneor more endogenous glycerol-regulating genes are downregulated to createyeast strains with altered glycerol production. In one embodiment,glycerol production in such yeast strains is downregulated in comparisonwith wild type yeast cell.

Pyruvate Formate Lyase (PFL)

The conversion of the pyruvate to acetyl-CoA and formate is performed bypyruvate formate lyase (PFL). In E. coli, PFL is the primary enzymeresponsible for the production of formate. PFL is a dimer of PflB thatrequires the activating enzyme PflAE, which is encoded by pflA, radicalS-adenosylmethionine, and a single electron donor. See Waks, Z., andSilver, P. A., Appl. Env. Microbiol. 75:1867-1875 (2009). Waks andSilver engineered strains of S. cerevisiae to secrete formate by theaddition of PFL and AdhE from E. coli and deletion of endogenous formatedehydrogenases and to produce hydrogen in a two-step process using E.coli. Waks and Silver, however, did not combine formate production withthe removal of glycerol formation, and the use of formate as analternate electron acceptor for the reduction of glycerol was notproposed or evaluated.

PFL enzymes for use in the recombinant host cells of the invention cancome from a bacterial or eukaryotic source. Examples of bacterial PFLinclude, but are not limited to, Bacillus licheniformis DSM13, Bacilluslicheniformis ATCC14580, Streptococcus thermophilus CNRZ1066,Streptococcus thermophilus LMG18311, Streptococcus thermophilus LMD-9,Lactobacillus plantarum WCFS1 (Gene Accession No. lp_2598),Lactobacillus plantarum WCFS1 (Gene Accession No. lp_3313),Lactobacillus plantarum JDM1 (Gene Accession No. JDM1_2695),Lactobacillus plantarum JDM1 (Gene Accession No. JDM1_2087),Lactobacillus casei bl23, Lactobacillus casei ATCC 334, Bifidobacteriumadolescentis, Bifidobacterium longum NCC2705, Bifidobacterium longumDJO10A, Bifidobacterium animalis DSM 10140, Clostridium cellulolyticum,or Escherichia coli. Additional PFL enzymes may be from the PFL1 family,the RNR pfl superfamily, or the PFL2 superfamily.

Examples of eukaryotic PFL include, but are not limited to,Chlamydomonas reinhardtii PflA1, Piromyces sp. E2, or Neocallimastixfrontalis, Acetabularia acetabulum, Haematococcus pluvialis, Volvoxcarteri, Ostreococcus tauri, Ostreococcus lucimarinus, Micromonaspusilla, Micromonas sp., Porphyra haitanensis, and Cyanophora paradoxa),an opisthokont (Amoebidium parasiticum), an amoebozoan (Mastigamoebabalamuthi), a stramenopile (Thalassiosira pseudonana (2)) and ahaptophyte (Prymnesium parvum), M. pusilla, Micromonas sp. O. tauri andO. lucimarinus) an amoebozoan (M. balamuthi), and a stramenopile (T.pseudonana). See Stairs, C. W., et al., “Eukaryotic pyruvate formatelyase and its activating enzyme were acquired laterally from afirmicute,” Mol. Biol. and Evol., published on-line on Feb. 3, 2011, athttp://mbe.oxfordjournals.org/.

Acetaldehyde/Alcohol Dehydrogenases

Engineering of acetaldehyde dehydrogenases, alcohol dehydrogenases,and/or bifunctional acetylaldehyde/alcohol dehydrogenases into a cellcan increase the production of ethanol. However, because the productionof ethanol is redox neutral, an acetaldehyde/alcohol dehydrogenaseactivity cannot serve as an alternative for the redox balancing that theproduction of glycerol provides to a cell in anaerobic metabolism. WhenMedina attempted to express an acetylaldehyde dehydrogenase, mhpF, fromE. coli in an S. cerevisiae gpd1/2 deletion strain, the strain did notgrow under anaerobic conditions in the presence of glucose as the solesource of carbon. Medina, V. G., et al., Appl. Env. Microbiol.76:190-195 (2010); see also EP 2277989. Rather, the anaerobic growth ofthe glycerol-deletion strain required the presence of acetic acid.However, an acetylaldehyde dehydrogenase has not been expressed incombination with PFL or with the recombinant host cells of theinvention. Additionally, replacing the endogenous acetylaldehydedehydrogenase activity with either an improved acetaldehydedehydrogenase or using a bifunctional acetaldehyde/alcohol dehydrogenase(AADH) can positively affect the in vivo kinetics of the reactionproviding for improved growth of the host strain.

Improving Conversion of Acetyl-CoA to Ethanol

To improve the conversion of acetyl-CoA to ethanol, endogenous yeastgenes can be replaced or complimented with either an improvedacetaldehyde dehydrogenase (e.g., from C. phytofermentans or othersource) to convert acetyl-CoA to acetaldehyde, or a bifunctionalacetaldehyde/alcohol dehydrogenase (AADH) to convert acetyl-CoA toacetaldehyde and acetaldehyde to ethanol. By engineering in one or moresuch enzymes, the in vivo kinetics of the conversion of acetyl-CoA toethanol can be increased, providing for improved growth of the hoststrain. The bi-functional alcohol/aldehyde dehydrogenase can come from avariety of microbial sources, including but not limited to E. coli, C.acetobutylicum, T. saccharolyticum, C. thermocellum, C. phytofermentans,Piromyces SP E2, or Bifidobacterium adolescentis.

When glycerol deletion strains are grown anaerobically, they are notcapable of growth or fermentation and cannot consume sugar duringglycolysis. However, if these glycerol deletion strains are complementedwith an AADH, the strains are able to grow with the supplementation ofacetate in the media.

AADH enzymes for use in the recombinant host cells of the invention cancome from a bacterial or eukaryotic source. Examples of bacterial AADHinclude, but are not limited to, Clostridium phytofermentans,Escherichia coli, Bacillus coagulans, Bacillus lentus, Bacilluslicheniformis, Bacillus pumilus, Bacillus subtilis, Bacteroidesamylophilus, Bacteroides capillosus, Bacteroides ruminocola, Bacteroidessuis, Bifidobacterium adolescentis, Bifidobacterium animalis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Bifidobacterium thermophilum, Lactobacillus acidophilus,Lactobacillus brevis, Lactobacillus buchneri (cattle only),Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii,Lactobacillus farciminis (swine only), Lactobacillus fermentum,Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum,Lactobacillus reuterii, Leuconostoc mesenteroides, Pediococcusacidilacticii, Pediococcus pentosaceus, Propionibacterium acidpropionici(cattle only), Propionibacterium freudenreichii, Propionibacteriumshermanii, Enterococcus cremoris, Enterococcus diacetylactis,Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis, orEnterococcus thermophiles.

Xylose Metabolism

Xylose is a five-carbon monosaccharide that can be metabolized intouseful products by a variety of organisms. There are two main pathwaysof xylose metabolism, each unique in the characteristic enzymes theyutilize. One pathway is called the “Xylose Reductase-XylitolDehydrogenase” or XR-XDH pathway. Xylose reductase (XR) and xylitoldehydrogenase (XDH) are the two main enzymes used in this method ofxylose degradation. XR, encoded by the XYL1 gene, is responsible for thereduction of xylose to xylitol and is aided by cofactors NADH or NADPH.Xylitol is then oxidized to xylulose by XDH, which is expressed throughthe XYL2 gene, and accomplished exclusively with the cofactor NAD⁺.Because of the varying cofactors needed in this pathway and the degreeto which they are available for usage, an imbalance can result in anoverproduction of xylitol byproduct and an inefficient production ofdesirable ethanol. Varying expression of the XR and XDH enzyme levelshave been tested in the laboratory in the attempt to optimize theefficiency of the xylose metabolism pathway.

The other pathway for xylose metabolism is called the “Xylose Isomerase”(XI) pathway. Enzyme XI is responsible for direct conversion of xyloseinto xylulose, and does not proceed via a xylitol intermediate. Bothpathways create xylulose, although the enzymes utilized are different.After production of xylulose both the XR-XDH and XI pathways proceedthrough the enzyme xylulokinase (XK), encoded on gene XKS1, to furthermodify xylulose into xylulose-5-phosphate where it then enters thepentose phosphate pathway for further catabolism.

Studies on flux through the pentose phosphate pathway during xylosemetabolism have revealed that limiting the speed of this step may bebeneficial to the efficiency of fermentation to ethanol. Modificationsto this flux that may improve ethanol production include a) loweringphosphoglucose isomerase activity, b) deleting the GND1 gene, and c)deleting the ZWF1 gene (Jeppsson et al., Appl. Environ. Microbiol.68:1604-09 (2002)). Since the pentose phosphate pathway producesadditional NADPH during metabolism, limiting this step will help tocorrect the already evident imbalance between NAD(P)H and NAD⁺ cofactorsand reduce xylitol byproduct. Another experiment comparing the twoxylose metabolizing pathways revealed that the XI pathway was best ableto metabolize xylose to produce the greatest ethanol yield, while theXR-XDH pathway reached a much faster rate of ethanol production(Karhumaa et al., Microb Cell Fact. 2007 Feb. 5; 6:5). See alsoInternational Publication No. WO2006/009434, incorporated herein byreference in its entirety.

In some embodiments, the recombinant microorganisms of the inventionhave the ability to metabolize xylose using one or more of the aboveenzymes.

Arabinose Metabolism

Arabinose is a five-carbon monosaccharide that can be metabolized intouseful products by a variety of organisms. L-Arabinose residues arefound widely distributed among many heteropolysaccharides of differentplant tissues, such as arabinans, arabinogalactans, xylans andarabinoxylans. Bacillus species in the soil participate in the earlystages of plant material decomposition, and B. subtilis secretes threeenzymes, an endo-arabanase and two arabinosidases, capable of releasingarabinosyl oligomers and L-arabinose from plant cell.

Three pathways for L-arabinose metabolism in microorganisms have beendescribed. Many bacteria, including Escherichia coli, use arabinoseisomerase (AraA; E.C. 5.3.1.4), ribulokinase (AraB; E.C. 2.7.1.16), andribulose phosphate epimerase (AraD; E.C. 5.1.3.4) to sequentiallyconvert L-arabinose to D-xylulose-5-phosphate through L-ribulose andL-ribulose 5-phosphate. See, e.g., Sa-Nogueira I, et al., Microbiology143:957-69 (1997). The D-xylulose-5-phosphate then enters the pentosephosphate pathway for further catabolism. In the second pathway,L-arabinose is converted to L-2-keto-3-deoxyarabonate (L-KDA) by theconsecutive action of enzymes arabinose dehydrogenase (ADH),arabinolactone (AL), and arabinonate dehydratase (AraC). See, e.g.,Watanabe, S, et al., J. Biol. Chem. 281: 2612-2623 (2006). L-KDA can befurther metabolized in two alternative pathways: 1) L-KDA conversion to2-ketoglutarate via 2-ketoglutaric semialdehyde (KGSA) by L-KDAdehydratase and KGSA dehydrogenase or 2) L-KDA conversion to pyruvateand glycolaldehyde by L-KDA aldolase. In the third, fungal pathway,L-arabinose is converted to D-xylulose-5-phosphate through L-arabinitol,L-xylulose, and xylitol, by enzymes such as NAD(P)H-dependent aldosereductase (AR), L-arabinitol 4-dehydrogenase (ALDH), L-xylulosereductase (LXR), xylitol dehydrogenase (XylD), and xylulokinase (XylB).These, and additional proteins involved in arabinose metabolism andregulation may be found athttp://www.nmpdr.org/FIG/wiki/rest.cgi/NmpdrPlugin/SeedViewer?page=Subsystems;subsystem=L-Arabinose_utilization,visited Mar. 21, 2011, which is incorporated by reference herein in itsentirety.

AraC protein regulates expression of its own synthesis and the othergenes of the Ara system. See Schleif, R., Trends Genet. 16(12):559-65(2000). In the E. coli, the AraC protein positively and negativelyregulates expression of the proteins required for the uptake andcatabolism of the sugar L-arabinose. Homologs of AraC, such asregulatory proteins RhaR and RhaS of the rhamnose operon, have beenidentified that contain regions homologous to the DNA-binding domain ofAraC (Leal, T. F. and de Sa-Nogueira, I., FEMS Microbiol Lett.241(1):41-48 (2004)). Such arabinose regulatory proteins are referred toas the AraC/XylS family. See also, Mota, L. J., et al., Mol. Microbiol.33(3):476-89 (1999); Mota, L. J., et al., J Bacteriol. 183(14):4190-201(2001).

In E. coli, the transport of L-arabinose across the E. coli cytoplasmicmembrane requires the expression of either the high-affinity transportoperon, araFGH, a binding protein-dependent system on the low-affinitytransport operon, araE, a proton symporter. Additional arabinosetransporters include those identified from K. marxianus and P.guilliermondii, disclosed in U.S. Pat. No. 7,846,712, which isincorporated by reference herein.

In some embodiments, the recombinant microorganisms of the inventionhave the ability to metabolize arabinose using one or more of the aboveenzymes.

The following embodiments of the invention will now be described in moredetail by way of these non-limiting Examples.

EXAMPLES Example 1: Expression of Fungal Lignocellulase SystemComponents in Yeast

In order to generate strains expressing these various enzymes, and inanticipation of co-expressing them, several promoter and terminatorpairs were created to use as expression vectors. The promoter terminatorpairs, and the enzyme types that were tested under their control arelisted in Table 3. Genes encoding various enzyme activities were clonedinto vector pMU1531 by standard molecular biology procedures (See e.g.Maniatis, “Molecular Cloning” Cold Spring Harbor Press). FIG. 2 gives aschematic of pMU1531 which was the backbone cloning vector used. Thisvector contains the ENO1 promoter and terminator from S. cerevisiae andthe URA3 and zeocin markers for use in yeast. It was subsequentlymodified to have the various promoter/terminator combinations listed inTable 3.

TABLE 3 Promoters and terminators used for expression of fungal andbacterial genes. # Promoter Terminator Genes expressed 1 ENO1 ENO1 EG1,EG2, EG3, xylanase (GH11 and GH10), xylosidase (GH43, GH3), completebacterial library 2 ENO1 PYK1 EG1 3 ADH1 PDC1 fungal GH10 xylanase, CpXyl10 (bacterial) 4 ADH2 CYC1 Beta-mannase, GH11 xylanase 5 ENO2 TDH3EG6 6 FBA1 PGI1 EG4 7 GPM1 TPI1 EG5 8 HXT7 PMA1 GH3 xylosidase, CIP1 9PDC1 ENO2 TfCel9A, GH74 xyloglucanase 10 PGI1 HXT7 GH10 xylanase 11 PMA1ADH1 EG2, 12 TDH3 GPM1 GH43 xylosidase 13 TPI1 FBA1 EG3 14 HXT2 ACT1GH27 (AGLI) 15 PFK1 HXT2 CE1 (AXE) 16 HXT3 PFK1 GH62 (AXH) 17 PFK2 HXT3CE1 (FAEA) 18 PYK1 PFK2 CE1 (FAEB) 19 TEF1 ADH2 SWO 20 ADH3 TEF1 GH2(beta-mannosidase) 21 TEF2 ADH3 GH67 (alpha-glucuronidase) 22 GND1 TEF2CIP2 23 ACT1 GND1 GH54 (ABF1) 24 TAL1 SOL1 alpha-expansin 25 TKL1 ADH5beta-expansin

TABLE 4 Fungal enzyme system components expressed in yeast. Cazy family/enzyme type/ synonym Activity Organism Accession # Strain # Plasmid #GH7B (EG1) Endoglucanase Aspergillus XP_747897 M1311 pMU1626 fumigatusGH7B (EG1) Endoglucanase Neosartorya XP_001257357 M1312 pMU1627 fischeriGH7B (EG1) Endoglucanase Aspergillus XP_001270378 M1313 pMU1628 clavatusGH7B (EG1) Endoglucanase Aspergillus XP_001217291 M1270 pMU1561 terreusGH7B (EG1) Endoglucanase Trichoderma ACZ34302 M1317 pMU1632longibrachiatum GH7B (EG1) Endoglucanase Penicillium XP_002152969 M1318pMU1633 marneffei GH7B (EG1) Endoglucanase Chaetomium XP_001229968 M1310pMU1625 globosum GH7B (EG1) Endoglucanase Neurospora XP_956431 M1271pMU1562 crassa GH7B (EG1) Endoglucanase Aspergillus oryzae BAA22589M1314 pMU1629 GH7B (EG1) Endoglucanase Thielavia AAE25067 M1315 pMU1630heterothallica GH7B (EG1) Endoglucanase Fusarium AAG09047 M1272 pMU1563oxysporum GH7B (EG1) Endoglucanase Humicola insolens 1DYM_A M1316pMU1631 GH7B (EG1) Endoglucanase Pyrenophora XP_001935476 M1319 pMU1634tritici-repentis GH7B (EG1) Endoglucanase Magnaporthe XP_370166 M1273pMU1564 grisea GH7B (EG1) Endoglucanase Fusarium XP_388429 M1274 pMU1565graminearum GH7B (EG1) Endoglucanase Hypocrea P07981 M1276 pMU1574jecorina GH5 (EG2) Endoglucanase Hypocrea P07982 M1138 pMU1400 jecorinaGH5 (EG2) Endoglucanase Chrysosporium RDH160 pRDH160 lucknowense GH5(EG2) Endoglucanase Polyporus BAF75943.1 RDH163 pRDH163 arcularius GH5(EG2) Endoglucanase Aspergillus BAB62317.1 RDH145 pRDH145 kawachii GH5(EG2) Endoglucanase Heterodera CAC12958.1 RDH146 pRDH146 schachtii GH5(EG2) Endoglucanase Orpinomyces sp. AAD04193.1 RDH148 pRDH148 GH5 (EG2)Endoglucanase Irpex lacteus BAD67544.1 RDH149 pRDH149 GH5 (EG2)Endoglucanase Chaetomium XP_001220409.1 RDH159 pRDH159 globosum GH5(EG2) Endoglucanase Aspergillus niger XP_001397982.1 RDH161 pRDH161 GH5(EG2) Endoglucanase Penicillium ABY28340.1 RDH162 pRDH162 decumbensGH12A Endoglucanase Trichoderma BAA20140 RDH164 pRDH164 (EG3) reeseiGH12A Endoglucanase Phanerochaete AAU12276 RDH167 pRDH167 (EG3)chrysosporium GH12A Endoglucanase Stachybotrys AAM77710 RDH165 pRDH165(EG3) echinata GH12A Endoglucanase Neosartorya XP_001261563 RDH166pRDH166 (EG3) fischeri GH12A Endoglucanase Chaetomium AAM77701 RDH168pRDH168 (EG3) brasiliense GH61A Endoglucanase Chaetomium EAQ86340 M1391pMU1746 (EG4) globosum GH61A Endoglucanase Aspergillus CAF31975 M1392pMU1747 (EG4) fumigatus GH61A Endoglucanase Humicola insolens CAG27577M1393 pMU1748 (EG4) GH61A Endoglucanase Neosartorya XP_001267517 M1394pMU1749 (EG4) fischeri GH61A Endoglucanase Thielavia ACE10231 M1418pMU1779 (EG4) terrestris GH45A Endoglucanase Chrysosporium ACH15008M1395 pMU1750 (EG5) lucknowense GH45A Endoglucanase ChaetomiumXP_001226436 M1420 pMU1753 (EG5) globosum GH45A Endoglucanase AcremoniumACE10216 M1421 YML only (EG5) thermophilum GH45A Endoglucanase Humicolainsolens CAB42307 M1396 pMU1751 (EG5) GH45A Endoglucanase ThielaviaCAH03187 M1418 pMU1779 (EG5) terrestris GH6 (EG6) EndoglucanaseChrysosporium AAQ38151 M1422 YML only lucknowense GH6 (EG6)Endoglucanase Magnaporthe EDJ97375 M1397 pMU1752 grisea GH6 (EG6)Endoglucanase Chaetomium EAQ84577 M1398 pMU1753 globosum GH6 (EG6)Endoglucanase Humicola insolens 1DYS_B M1399 pMU1754 GH6 (EG6)Endoglucanase Neurospera XP_957415 M1400 pMU1755 crassa GH74AXyloglucanase Trichoderma AAP57752 M1423 YML only (EGL6) reesei GH74AXyloglucanase Aspergillus niger AAK77227 M1424 YML only (EGL6) GH74AXyloglucanase Aspergillus BAA29031 M1425 YML only (EGL6) aculeatus GH74AXyloglucanase Neosartorya XP_001261776 M1426 YML only (EGL6) fischeriGH11 Endoxylanase Chaetomium CAD48749 RDH170 pRDH170 thermophilum GH11Endoxylanase Trichoderma ABK59833 RDH169 pRDH169 reesei (syntheticversion) GH11 Endoxylanase Trichoderma ABK59833 RDH182 pRDH182 reesei(native version) GH10 Endoxylanase Chrysosporium AAQ38147 RDH183 pRDH183lucknowense GH10 Endoxylanase Aureobasidium BAE71410 RDH171 pRDH171pullulans GH3 beta-xylosidase Aspergillus niger XP_001389416 RDH181pRDH181 GH3 beta-xylosidase Aspergillus CAA73902 RDH179 pRDH179 nidulansGH43 beta-xylosidase Cochliobolus AAC67554 RDH175 pRDH175 (BXL1)carbonum GH43 beta-xylosidase Penicillium BAC75546 RDH176 pRDH176 (BXL1)herquei GH43 beta-xylosidase Pyrenophora XP_001940956 RDH177 pRDH177(BXL1) tritici-repentis MAN1 beta-mannase Aspergillus AAA67426 pMU1903(endo-enzyme) aculeatus GH2 beta- Aspergillus niger Q9UUZ3 M1491 pMU1912mannosidase (exo-enzyme) GH2 beta- Aspergillus BAA29029 M1492 pMU1913mannosidase aculeatus (exo-enzyme) GH2 beta- Neosartorya XP_001258000M1493 pMU1914 mannosidase fischeri (exo-enzyme) GH67 alpha- TrichodermaCAA92949 M1494 pMU1915 glucuronidase reesei GH67 alpha- Aspergillusniger CAC38119 M1547 YML only glucuronidase GH67 alpha- TalaromycesAAL33576 M1549 YML only glucuronidase emersonii CE1 (AXE)acetylxylanesterase Aspergillus niger XP_001395572 M1513 pMU1933 CE1(AXE) acetylxylanesterase Trichoderma Q99034 M1512 pMU1932 reesei CE1(AXE) acetylxylanesterase Neosartorya XP_001262186 M1514 pMU1934fischeri GH27 (AGLI) alpha- Trichoderma CAA93244 M1550 YML onlygalactosidase reesei (AGLI) GH54 arabinofuranosidase Aspergillus nigerAAA93264 M1511 pMU1930 (ABF1) GH62 (ABF2, arabinofuranosidase,Trichoderma AAP57750 M1483 pMU1904 AXHA) 1,4-beta- reesei D-arabinoxylanarabinofuranohydrolase GH62 (ABF2, arabinofuranosidase, ChaetomiumXP_001223478 M1479 pMU1885 AXHA) 1,4-beta- globosum D-arabinoxylanarabinofuranohydrolase GH62 (ABF2, arabinofuranosidase, Aspergillusniger XP_001389998 M1481 pMU1890 AXHA) 1,4-beta- D-arabinoxylanarabinofuranohydrolase SWO Swollenin Penicillium ACH57439 M1471 pMU1876(expansin) decumbens SWO Swollenin Neosartorya XP_001257521 M1472pMU1877 (expansin) fischeri SWO Swollenin Talaromyces EED19018 M1473pMU1878 (expansin) stipitatus SWO Swollenin Trichoderma CAB92328 M1515pMU1931 (expansin) reesei CIP1 Unknown Trichoderma AAP57751 M1484pMU1905 reesei CIP1 Unknown Chaetomium XP_001228455 M1485 pMU1906globosum CIP1 Unknown Magnaporthe XP_365869 M1486 pMU1907 grisea CIP2glucuronyl Trichoderma AAP57749 M1482 pMU1891 esterase reesei CIP2glucuronyl Chaetomium XP_001226041 M1474 pMU1879 esterase globosum CIP2glucuronyl Aspergillus XP_751313 M1480 pMU1886 esterase fumigatus alpha-alpha-expansin Populus alba BAB39482 M1488 pMU1909 expansin alpha-alpha-expansin Vitis lubrusca BAC66697 M1487 pMU1908 expansinbeta-expansin beta-expansin Triticum aestivum AAS48881 M1490 pMU1911beta-expansin beta-expansin Eucalyptus AAZ08315 M1489 pMU1910 globulusCE1 (FAEA) Feruoyl esterase Aspergillus niger XP_001393337 M1475 pMU1880(FAEA) CE1 (FAEA) Feruoyl esterase Aspergillus XP_001211092 PleasepMU1884 (FAEA) terreus provide CE1 (FAEB) Feruoyl esterase TalaromycesEED17739 M1476 pMU1881 (FAEB) stipitatus CE1 (FAEB) Feruoyl esteraseChaetomium XP_001228412 M1477 pMU1882 (FAEB) globosum

Example 2: Characterizing the Expression and Activity of AuxillaryCellulases

Following strain construction, strains expressing the fungal EG1candidates were grown in 50 mL shake flask cultures with 100 ug/mLzeocin and tested for activity on CMC and avicel. FIG. 3 demonstratesthat several active EG1s were found and that several were superior inactivity to the comparable enzyme previously used (Trichoderma reeseiEG1, M1276). From these data, the top 6 candidates were selected basedon activity on avicel for further testing on PHW (FIGS. 4 and 5).

The PHW assay was carried out with a pretreated wood substrate (MS149),both in the presence and absence of yeast made, purified CBH1 and CBH2(2 mg/g of each), and Novozyme 188 BGL. 2 mL of supernatant was usedfrom each EG1 expressing strain in the assay. A strain expressing TrEG2from the same plasmid was again used as a control. The results fromthese assays can be found in FIGS. 4 and 5. Several EG1s showed theability to act with CBH1 and CBH2 to increase hydrolysis, although notto the level that TrEG2 is capable of. Similarly, several EG1s showedthe ability to release glucose from PHW in the presence of Novozymes 188(a crude beta-glucosidase preparation containing several activitiesbeyond BGL), and several also showed more xylose release than just thestrain background alone.

Given the strong performance of M1311 in CMC, avicel and PHW assays, andthe fact that it has a native CBD, the Aspergillus fumigatus enzyme waschosen as the best EG1 candidate.

In order to investigate other EG2-type endoglucanases and to investigateEG3-type endoglucanases for enhancement of current cellulase expressionconfigurations. The choice of additional cel5 sequences was based onsequences with relatively good homology to the T. reesei eg2 orAspergillus kawachii egA, the most successfully expressed cel5 genesfrom the first round of testing. The choice of cel12 sequences to betested was based on sequences with relatively good homology to the T.reesei eg3 although sequences with homology greater than 95% weredisregarded. Table 4 indicates the genes chosen for synthesis as well asthe designation of the expression vector. All the genes were clonedunder control of the ENO1 promoter/terminator using the pMU1531expression plasmid.

The plasmids were all transformed to S. cerevisiae M0509 (anindustrially hearty strain expressing xylose isomerase) using YPDcontaining 250 μg/ml zeocin as selective medium and transformants wereconfirmed with PCR. Along with the reference strain (containing pMU1531)and a strain expressing the T. reesei eg2 (pRDH180), the eg2/eg3expressing strains were tested for activity on avicel and CMC. Thestrains were grown in YPD or double strength SC medium (3.4 g/L YNB; 3g/L amino acid pool; 10 g/L ammonium sulfate; 20 g/L glucose) that wasbuffered to pH 6 (20 g/L succinic acid; 12 g/L NaOH, set pH to 6 withNaOH). Glucose was added after autoclaving of the other components froma 50% glucose stock solution. Zeocin was added to a final concentrationof 100 μg/ml for liquid cultures. 10 mL cultures in 125 mL erlenmeyerflasks were grown at 30° C. for three days (YPD) or four days (SC).

Three flasks were inoculated for each strain. After incubation, sampleswere taken for gel analysis, protein determination and activitymeasurement. After centrifugation of the samples, 12 μl of each wastaken, added to 5 μl of protein loading buffer and boiled for 5 minutes.The samples were subsequently loaded on a 10% SDS-PAGE and separated,followed by silver staining (FIG. 6).

From the gel it appeared that not all strains produced a visible band inthe expected size range (see Table 5 for predicted sizes). The T.r.EG2appeared as a band of about 55 kDa. As it was predicted to beapproximately 44 kDa, the extra weight may represent hyperglycosylation.The EG2s of C. lucknowense, A. niger, and P. decumbens were also visiblein the same approximate size range with the P. decumbens product beingslightly smaller at ˜50 kDa. From the gel it appeared that far more C.lucknowense EG2 protein was produced compared to the other EG2s. FromFIG. 6B it was clear that there were no visible bands for the S.echinata or P. chrysosporium eg3 gene products. The T. reesei, N.fischeri and C. brasiliense eg3 gene products were visible as 30, 25 and35 kDa bands, respectively. Again, the extra weight may representhyperglycosylation. However, the N. fischeri Eg3 was found to be at orvery near to its predicted size—this protein contains no putativeN-glycosylation sites.

To screen for EG activity, 5 μl of the cultures used for quantitativeassays were spotted on SC^(−URA) plates containing 0.2% of either CMC orbarley-β-glucan (FIG. 7). Two CMC containing plates were made andstained after 3 or 24 hours. As can be seen from FIG. 7 the T.r.eg2expressing strain (180) yielded very good clearing zones on bothsubstrates. The other eg2 expressing strains also showed good clearingzone formation along with the strains expressing EG3's from T. reesei(164), S. echinata (165), N. fischeri (166) and C. brasiliense (168).The N. fischeri eg3 expressing strain (166) consistently yielded largerclearing zones than the other EGs on the plate assays. Due to thesmaller size of this protein (FIG. 6B) and apparent lack ofglycosylation this enzyme may have superior diffusion qualities in thismedia.

All strains were tested for activity using the high-throughput avicelconversion method as prescribed. Activity on CMC was determined with asimilar assay while omitting the Novozyme 188 and starting with 1% CMC.The DNS used for the assay procedure contained phenol. Activity datafrom strains grown on YPD and SC can be seen in FIG. 8.

From the activity data it would appear that the strain expressing T.reesei eg2 (pRDH180) produced the highest levels of secreted activity.The EG2 from C. lucknowense displayed the next best activity on bothsubstrates. The T. reesei EG3 and N. fischeri EG3 appear to be thesuperior enzymes for yeast expression from this group (cel12, willsubsequently be tested on PHW).

Strain M0509 was also transformed with 2 um plasmids containing EG4s,EG5s, EG6s, and xyloglucanases (GH74/XG). These strains were thenspotted on YNB plates with CMC, grown overnight at 30 degrees andstained with Congo red to check for activity of the cloned gene (Datafor some of the strains shown in FIG. 9). The EG4 genes showed only weakactivity on CMC, while both EG5 candidates showed large clearing zones,and all EG6s showed intermediate clearing zone size. The XG candidatesall showed very small clearing zones on CMC. All enzyme types gavefunctional candidates.

The candidates were also tested for activity in the PHW assay in thepresence of other enzymes. Purified, yeast made CBH1, CBH2, EG2, and BGLwere used as partners for the assay loaded at a 4 mg enzyme protein pergram of solids, and a 40%:40%:15%:5% (by mass) mixture (FIG. 10). Ascontrols, M0509 supernatant (negative) or M1179 supernatant (positivecontrol strain expressing CBH1, CBH2, EG2, and BGL) were used.

The data in FIG. 10 demonstrate that addition of EG4 (from Chaetomiumglobosum or Neosartorya fischeri) or EG5 (from Chrysosporiumlucknowense) can increase the hydrolysis of a 4 mg/g loading of CBHs,EG2, and BGL. When compared to loading an additional dose of CBH1, CBH2,EG2, and BGL (1179 supernatant), EG4 and EG5 give an increase in glucoserelease, although this difference does not appear to be statisticallysignificant based on data from the glucose assay kit. Regardless,candidates for these 3 categories have been obtained, although severalmore remain to be screened.

The XG candidates, and several EG4, 5, and 6 candidate genes along withthe best candidates from the previous round of assays for EG4, 5, and 6were used in a PHW assay (FIG. 11). The results indicate that several ofthe enzymes have activity on PHW. The EG4s from C. globosum and T.terrestris both gave an increase in glucose release relative to thenegative control and relative to the strain expressing T. reesei EG2.The same was true for the C. globosum EG5, and the N. crassa EG6. The XGcandidates showed only a very minor increase in reaction over thecontrol strain, with the N. fischerii XG appearing to be the best.

Example 3: Cloning and Expression of 5 Synthetic Xylanases and 5Synthetic Xylosidases in S. cerevisiae

Xylanases and xylosidases were examined for expression in yeast in orderto broaden the enzymatic activity spectrum of the yeast madelignocellulolytic system. Xylanases were selected from the publicdatabases and their functional expression in yeast was tested onsubstituted xylans. Xylosidases were selected based on homology to A.niger xlnD (a GH family 3 enzyme) and to include xylosidases from GHfamily 43. Table 5 (condensed version of Table 4) indicates the geneschosen for synthesis as well as the designation of the expressionvector. All the genes were cloned under control of the ENO1promoter/terminator using the pMU1531 expression plasmid. The plasmidswere all transformed to S. cerevisiae M0509 and transformants wereconfirmed with PCR.

TABLE 5 Xylanase and xylosidase encoding genes expressed in S.cerevisiae. GH Expression Theoretical size Organism & Gene: Family:plasmid: (kDaa) Xylanases: T. reesei xyn2 (native sequence) 11 pRDH18221.0 T. reesei xyn2 (synthetic) 11 pRDH169 21.0 Chaetomium thermophilum11 pRDH170 27.8 xyn11A Aureobasidium pullulans var. 10 pRDH171 39.9melanigenum xyn10 Cryptococcus albidus xylanase 10 pRDH172 35.8Aspergillus niger xylanase D 43 pRDH174 35.4 Xylosidases: Aspergillusniger xlnD - native 3 pRDH181 86.7 sequence (S.c.MFα secretion signal)Cochliobolus carbonum 43 pRDH175 36.8 β-xylosidase Penicillium herqueixylosidase 43 pRDH176 37.4 Pyrenophora tritici-repentis β- 43 pRDH17736.9 xylosidase Aspergillus nidulans xylosidase 3 pRDH179 87.1

Along with the reference strain (containing pMU1531), a strainexpressing the native sequence of T.r.xyn2 (pRDH182) and a strainexpressing the native sequence of A.n.xlnD (pRDH181), thexylanase/xylosidase expressing strains were tested for activity on 1%birchwood glucuronoxylan (Roth) and pNP-xylopyranoside (pNPX). Thestrains were grown in YPD or buffered double strength SC medium (pH 6).Zeocin was added to a final concentration of 100 μg/mL for liquidcultures. 10 mL Cultures in 125 mL Erlenmeyer flasks were incubated at30° C. for three days (YPD) or four days (SC). Three flasks wereinoculated for each strain. After incubation, samples were taken for gelanalysis, protein determination and activity measurement. Aftercentrifugation of the samples, 12 μL of each was taken, added to 5 μL ofprotein loading buffer and boiled for 5 minutes. The samples weresubsequently loaded on a 10% SDS-PAGE and separated, followed by silverstaining (FIG. 12).

From the gel it appeared that not all strains produced a visible band inthe expected size range (see Table 5 for predicted sizes). (A) TheT.r.XYN2 appeared as a band of about 21 kDa as predicted. The Chaetomiumthermophilum XYN11A is visible as a faint band of about 36 kDa, largerthan the expected 27.8 kDa. The Aureobasidium pullulans XYN10 is visibleas a prominent band at ˜50 kDa. The Cryptococcus albidus and Aspergillusniger xylanases are also visible as bands slightly larger than predictedbut these gene products yielded no activity in liquid assays (FIG. 14).The increased sizes of the secreted enzymes can likely be explained as aresult of hyperglycosylation. (B) A large smear at >90 kDa may representheterogeneously glycosylated forms of the A. niger XLND xylosidase. TheCochliobolus carbonum, Penicillium herquei, and Pyrenophoratritici-repentis xylosidases are present as 45, 50 and 55 kDa bands(slightly smeared), larger than the predicted ˜37 kDa also indicatinglikely hyperglycosylation.

To screen for xylanase activity, 5 μL of the cultures used forquantitative assays were spotted on an SC^(−URA) plate containing 0.2%RBB-xylan and incubated for 24 hours (FIG. 13). As can be seen from thefigure, the T.r.xyn2 expressing strain (RDH182) yielded a very goodclearing zone whereas the reference strain did not. Of the otherxylanase expressing strains Chaetomium thermophilum xyn11A andAureobasidium pullulans xyn10 yielded clearing zones but none of theother strains produced a visible clearing zone.

All strains were tested for activity on birchwood glucuronoxylan (Roth)and pNP-xylopyranoside (pNPX). Xylanase assays were performedessentially as described in La Grange et al. (1996, Appl. Environ.Microbiol. 62, 1036-1044). Reactions were miniaturized for use in a96-well PCR plate. 5 μL supernatant was added to 45 μL 1% glucuronoxylanand incubated at 35° C. for 5 minutes. Reactions were stopped by adding75 μL DNS before heating at 99° C. for 5 minutes. A standard curve wasset using xylose. Xylosidase assays were performed in the same manner asfor β-glucosidase assays (see above protocol) but with pNPX as substrateat pH5, 35° C. for 2-5 minutes depending on the activity. Activity datafrom strains grown on YPD and SC can be seen in FIG. 14.

From the activity data it would appear that the strain expressing thenative T.r.xyn2 (pRDH182) produced the highest levels of secretedxylanase activity. It was surprising that the strain containing a codonoptimized version of this gene (sequence verified) displayed no secretedactivity. The GH family 11 xylanase encoded by Chaetomium thermophilumxyn11A did give notable activity, however, far less than that generatedby the strain expressing native T.r.xyn2. The strain expressingAureobasidium pullulans xyn10 (GH family 10) also yielded appreciableactivity. This is particularly encouraging as it is known that family 10xylanases often have only 10% of the specific activity of GH family 11enzymes. However, family 10 xylanases are less restricted in theiraction by side chain substitutions on the xylan backbone. Somewhatsurprisingly, the GH family 43 xylosidases encoded by the genes fromCochliobolus carbonum and Pyrenophora tritici-repentis gave substantialxylanase activity. These enzymes are also classed as “exo-xylanases” andit will be very interesting to see how they interact with other xylandegrading enzymes. The strains producing these two enzymes alsodisplayed far greater xylosidase activity on pNPX than the strainexpressing native A.n.xlnD. Furthermore, the strain expressing nativeA.n.xlnD secreted only about 36% of the total xylanase activity itproduced when grown in YPD whereas 76% and 99% of the C. carbonum and P.tritici-repentis heterologous xylosidases were secreted. The secretedxylosidase activities of the strains producing C. carbonum and P.tritici-repentis xylosidases in YPD were respectively 3.3 and 6.9 foldhigher than the secreted activity of the strain expressing nativeA.n.xlnD.

An assay assessing synergy of the best xylanases and xylosidasesidentified is shown in FIG. 15. Birchwood glucuronoxylan (5% in 50 mMNaOAc, pH5) was prepared and 400 μL aliquots were placed in a deep wellplate. Subsequently, supernatants of SC-grown yeast strains were addedas follows:

1.100 μl supernatant of REF strain

2. 50 μl supernatant of REF strain, 50 μl supernatant of RDH182 strain(T.r.xyn2)

3. 50 μl supernatant of REF strain, 50 μl supernatant of RDH171 strain(A.p.xyn10)

4. 50 μl supernatant of REF strain, 50 μl supernatant of RDH177 strain(P.tr.xld)

5. 50 μl supernatant of RDH182 strain (T.r.xyn2), 50 μl supernatant ofRDH177 strain (P.tr.xld)

6. 50 μl supernatant of RDH171 strain (A.p.xyn10), 50 μl supernatant ofRDH177 strain (P.tr.xld)

The mixtures were shaken on a microtiter plate shaker at 1000 rpm, 35°C. for 22 hours. DNS assays were performed to ascertain the amounts ofreducing sugar formed (FIG. 15). From this result it would seem thatthere was a synergistic effect when the xylanases and the xylosidasewere mixed. The activity of the T.r.XYN2 and P.tr.XLD mix was 1.24 timesmore than the sum of the activities separately. The activity of theA.p.XYN10 and P.tr.XLD mix was 1.9 times more than the sum of theactivities of those supernatants separately. To analyze the releasedsugars, 5 μL of each reaction and standards were spotted on a silicacoated thin layer chromatography (TLC) plate and separated with andeluant consisting of isopropanol: ethanol: water (7:1:2). The plate wasthen developed by dipping it in a mixture of 5% H₂SO₄ (made in ethanol)and heating in a 180° C. oven (FIG. 16). The action of the xylanases(lanes 2 and 3) yielded small amounts of xylotriose and more significantamounts of xylobiose. The xylosidase from P. tritici-repentis released asmall amount of xylose from xylan (lane 4). Mixtures of theheterologously produced xylanases with the xylosidase yieldedsignificant amounts of xylose (lanes 5 and 6) with no visiblexylo-oligos remaining in these reactions. These reactions will befurther analysed with HPLC analysis. The results presented in FIGS. 15and 16 show that the promising xylanases and xylosidases identified inthis study can synergise and yield the desired product namely xylose.

Derivatives of M0509 expressing the T. reesei Xyn2 (xylanase, pRDH182),and the P.t.r. GH43 xylosidase (xylosidase, pRDH177), or both theenzymes (pMU1819 below) were created. A cassette to integrate bothenzymes was created so that both enzymes could be integrated at the rDNAlocus. (FIG. 40). Selection was carried out via the natMX marker. Theability of the three strains to utilize xylan was tested by cultivatingthem in media containing yeast extract (1%), peptone (2%), glucose (2%),and xylan (5%). For each strain the percentage of the xylan that couldbe converted to ethanol in this test is shown in FIG. 39. The resultsdemonstrate the synergy between the two enzymes as well as the abilityto create a strain that can directly convert xylan to ethanol.

Example 4: Screening of Fungal Accessory Enzymes

Assays for arabinofuranosidase activity and esterase activity werecarried out to assess whether any of the accessory enzymes werefunctional. The arabinofuranosidase assay was carried out as follows:Substrate (1 mM 4-nitrophenyl-L-arabinofuranoside (Sigma #N-3641)) wasmade up in 50 mM citrate buffer pH 5.4 and preheated to 35 C. 20 ul ofyeast supernatant plus 180 ul of substrate was added to 96 well plate,and incubated at 35 degrees for 30 minutes. The reaction was stopped byadding 100 ul of 1M Na₂CO₃ and an OD measurement was taken at 405 nM.Zoomerase (lul) at a concentration of 177 ug/ul was added in a total of20 ul citrate buffer. The esterase activity assay was carried out asfollows: A 200 mM stock of substrate (4-Nitrophenol Acetate-SigmaN-8130) was made up in DMSO; 50 ul of this stock was added to 10 mls ofcitrate buffer pH 5.4 to make a 1 mM final concentration. 50 ul ofsupernatant to be tested was added to a 96 well flat bottom plate plus100 ul of substrate solution. The reaction was incubated at 35 degreesfor 30 minutes and the OD at 410 nm was taken.

FIGS. 17 and 18 show the results for the assays that were carried out.Only the Abfb gene from A. niger showed activity on the syntheticsubstrate pNPA. This confirms expression of this gene, which has beenpreviously expressed in yeast (Crous et al. 1996), in our strain. TheGH62 arabinofuranosidase candidates did not show activity on thissubstrate, which could be due to poor expression, or an inability tocleave the substrate. Several genes were shown to have activity on thesynthetic substrate p-Nitrophenol-actetate (FIG. 18). Candidates forboth types of feruoyl esterases (FAEA and FAEB), as well as one of theacetyl xylan esterases (AXE) were shown to be active.

PHW assays were set up to screen several accessory components and assesstheir impact in the presence of other yeast made enzymes. FIG. 19 showsthe results of the first screen, which demonstrate that both theNeosartorya fischeri and the Trichoderma reesei AXE genes expressed inM0544 yield increased xylan and glucan hydrolysis from unwashedpretreated hardwood substrate (MS630). In fact, without the AXEspresent, there is no measurable release of xylose from this substrateusing the yeast made xylanase and xylosidase. The hydrolysis of thexylan in MS630 should result in ˜1.8 g/L xylose release in this assay,thus the ˜1.4 g/L observed is about 77% of the total available, anincrease of 25% over the control. Glucose hydrolysis was increased by˜25% by the presence of the N.f. AXE.

FIG. 20 shows the results of attempting combinations of enzymes onunwashed MS630 (a pretreated hardwood substrate). A couple ofinteresting results can be observed. One is that in the presence ofzoomerase (1 mg/g) the accessories are having only a small impact onhydrolysis glucan in MS630 at the loadings tested. However, xylanhydrolysis is substantially increased by the presence either the N.f.AXE (acetylxylanesterase) or the Tr. AXE, with the best combinationsyielding ˜90% conversion. In the absence of zoomerase these enzymesincreased the hydrolysis of both glucan and xylan. Additionally,reducing the amount of AXE and simultaneously increasing the loading ofyeast made xylanase and xylosidase increased the rate of xylose release,indicating that these enzymes are the rate limiting ones needed athigher expression levels. The best combination of enzymes withoutzoomerase yielded ˜72% conversion of the xylan to xylose.

Example 5: Testing Endoglucanases for Possible Xylanase Activity

It was shown previously that fungal and bacterial xylanases of GH10 andGH11 produce ethylxylanopyranoside (EXP) during fermentation. In orderto find xylanases that do not produce EXP several fungal and bacterialenzymes belonging to different GH families were tested for xylanaseactivity. Enzymes from GH families 5, 7, 8, 10, 11, 12, 16, 26, 43, 44,and 51 were screened for activity on xylan as members of these familieshave been reported to contain some xylanase activity. Cultures weregrown in YPD for 72 h and the supernatants were evaluated on thebirchwood xylanase assay (FIG. 21).

FIG. 21 demonstrates that BC 60 displayed significant xylanase activity,and also, the strains containing a fungal GH10 xylanase from A.pullulans (M1379), and two GH7 EG1's from Aspergillus fumigatus (M1311)and Trichoderma longibrachiatum (M1317) did have activity on birchwoodxylan, although it was less than BC60 and T. reesei xyn2 (Con5).

Example 6: Expression of Bacterial Lignocellulolytic Enzyme SystemComponents in Yeast

Several potential bacterial donors of lignocellulolytic enzymes arelisted in Table 6, with preference given to mesophilic organisms withnoncomplexed cellulases. At the same time bacteria from different groups(aerobic vs. anaerobic and meso vs. thermo) were selected, to providediversity. Also, preferred donors were chosen if the functionalexpression of their genes in yeast was previously reported (Thermobifidafusca, Cellulomonas fimi, Clostridium phytofermentans, etc.). GC contentof bacterial genomes also influenced the choice of donor. The preferencewas given to the organisms with GC content that is not too far from S.cerevisiae GC content—38% (see Table 6), although the organisms withhigh GC content also were not completely ruled out based on successfulexpression in yeast of native cel9A from T. fusca that has 67.5 GCcontent.

Table 7 gives the full list of the bacterial genes screened forexpression in yeast. All the genes except those indicated weresuccessfully amplified by PCR from genomic DNA and transformed intoyeast strain together with the 2μ vector backbone for cloning via yeastmediated ligation. The enzymes not cloned from genomic DNA wereavailable as codon optimized versions.

TABLE 6 Characteristics of various bacterial donors of cellulolyticenzymes, DBM-disulphide bonds machinery. GC Oxygen Growth GrowthCellulase con- Organism relation temp. pH system tent DBM StreptomycesAerobe Meso 7 Non- 70.7 + avermitilis complexed, cell free Saccharo-Aerobe Meso 7.6 Non- 45.8 + phagus complexed, degradans cell freeBacillus Facult. Meso 6.8 Non- 43.5 + subtilis complexed, cell freeClostridium Anaerobe Meso 7.5 Combined 37.4 + cellulolyticum ClostridiumAnaerobe Meso 7 Non- 35.3 + phyto- complexed, fermentans cell freeThermobifida Aerobe Thermo 7.4 Non- 67.5 + fusca complexed, cell freeClostridium Anaerobe Thermo 6.7 Combined 39 − thermocellum

TABLE 7 Bacterial genes screened for expression in Saccharomycescerevisiae. In certain figures and examples, BC # designates the enzymeused in that experiment. Organism Activity GHF Gene or locus taq ProteinID BC # MESOPHILES Aerobes Streptomyces Exo  6 1,4-beta- NP_821732.1 1avermitilis cellobiosidase guxA1 Streptomyces Exo  6 1,4-beta-NP_823029.1 2 avermitilis cellobiosidase guxA2 Streptomyces exo/endo 481,4-beta- NP_823031.1 3 avermitilis cellobiosidase guxA3 Streptomycesendoglucahase/ 12 endo-1,4-beta- NP_821730.1 4 avermitilis xylanase?glucanase celA1 Streptomyces Endo endo-1,4-beta- NP_823030.1 5avermitilis glucanase celA2 Streptomyces endo endo-1,4-beta- NP_823032.16 avermitilis glucanase celA3 Streptomyces endoglucahase/ 12endo-1,4-beta- NP_823744.1 7 avermitilis xylanase? glucanase celA4Streptomyces endo  6 endo-1,4-beta- NP_826394.1 8 avermitilis glucanaseStreptomyces endo  6 endo-1,4-beta- NP_828072.1 9 avermitilis glucanasecelA5 Streptomyces endoxylanase 10 beta-1,4-xylanase NP_823272.1 10avermitilis Streptomyces endoxylanase 10 beta-1,4-xylanase NP_826161.111 avermitilis Streptomyces xylanase/ 43 xylanase NP_827548.1 12avermitilis xylosidase ? Streptomyces xylanase/xylosidase ? 43endo-1,4-beta- NP_827557.1 13 avermitilis xylanase xynD Streptomycesxylosidase 39 1,4-beta-xylosidase NP_822628.1 14 avermitilis xynB1Streptomyces xylanase/ 43 beta-xylosidase NP_823285.1 15 avermitilisxylosidase ? Streptomyces xylosidase/  3 1,4-beta-xylosidase NP_826159.116 avermitilis glucosidase? xynB2 Streptomyces xylosidase 391,4-beta-xylosidase NP_827745.1 17 avermitilis xynB3 Streptomycesbeta-glucosidase  1 beta-glucosidase NP_822977.1 18 avermitilis bglC1Streptomyces beta-glucosidase  1 beta-glucosidase NP_826430.1 19avermitilis bglC2 Streptomyces beta-glucosidase  1 beta-glucosidaseNP_826775.1 20 avermitilis bglC3 Streptomyces Acetyl xylan AXE1NP_822477.1 21 avermitilis esterase Streptomyces Acetyl xylan AXE1NP_822632.1 22 avermitilis esterase Streptomyces arabinofuranosidase/ 43abfA NP_822218.1 23 avermitilis xylanase Streptomycesarabinofuranosidase/ abfB NP_822290.1 24 avermitilis xylanaseStreptomyces arabinofuranosidase abfA NP_826920.1 25 avermitilisStreptomyces arabinofuranosidase/ abfB BAC74043.1 26 avermitilisgalactosidase Streptomyces arabinofuranosidase SAV_6756 BAC74467.1 27avermitilis Streptomyces galactosidase agaA1 BAC68338.1 28 avermitilisStreptomyces galactosidase agaA3 BAC68787.1 29 avermitilis Streptomycesgalactosidase agaB2 BAC69185.1 30 avermitilis Saccharophagus Endo  5?Sde_2993 YP_528462.1 31 degradans 2-40 Saccharophagus Endo  5? Sde_2996YP_528465.1 32 degradans 2-40 Saccharophagus Endo  5? Sde_3023YP_528492.1 33 degradans 2-40 Saccharophagus Endo  5 cel5A ABD82260.1 34degradans 2-40 Saccharophagus Endo  5 cel5E ABD82186.1 35 degradans 2-40Saccharophagus Endo  5 cel5F ABD80834.1 36 degradans 2-40 SaccharophagusEndo  5 cel5J ABD81754.1 37 degradans 2-40 Saccharophagus Endo  9 cel9AABD79898.1 38 degradans 2-40 Saccharophagus beta-glucosidase  3 ced3AABD81757.1 39 degradans 2-40 Saccharophagus beta-glucosidase  3 ced3BABD79509.1 40 degradans 2-40 Saccharophagus beta-glucosidase  1 bgl1AABD82858.1 41 degradans 2-40 Saccharophagus beta-glucosidase  1 bgl1BABD80656.1 42 degradans 2-40 Saccharophagus Cellobiose 94 Cep94AABD80580.1 43 degradans 2-40 phosphorylase Saccharophagus Cellodextrin94 Cep94B ABD80168.1 44 degradans 2-40 phosphorylase Saccharophagusmannanase Sde_0509 YP_525985.1 45 degradans 2-40 Saccharophagusmannosidase  2 Sde_0169 YP_525645.1 46 degradans 2-40 FacultativeAnaerobes Bacillus subtilis synergy with expansin exlX CAB13755.1 47endo/exo Bacillus subtilis endo/exo? endo-1,4-beta- CAB13696.2 48glucanase eglS Bacillus subtilis endo/exo 30 endo-xylanase xynCCAB13698.1 49 xlylanase? Bacillus subtilis endo/exo 43 endo-1,4-beta-CAB13699.1 50 xlylanase? xylanase xynD Bacillus subtilis endo xlylanase11 endo-1,4-beta- CAB13776.1 51 xylanase xynA Bacillus subtilisxylanase/ 43 xylan beta-1,4- CAB13642.2 52 xylosidase ? xylosidase xynBAnaerobes Clostridium Exo/Endo  9 Cphy_3367 YP_001560459.1 53phytofermentans Clostridium Exo/Endo 48 Cphy_3368 YP_001560460.1 54phytofermentans Clostridium Endo  5 Cphy_2058 YP_001559165.1 55phytofermentans Clostridium Endo  5 Cphy_3202 celulase B YP_001560295.156 phytofermentans Clostridium Endo  5 Cphy_1163 YP_001558280.1 57phytofermentans Clostridium beta-glucosidase  3 Cphy_3329 YP_001560421.158 phytofermentans Clostridium beta-glucosidase  3 Cphy_1125YP_001558242.1 59 phytofermentans Clostridium xylanase 10 Cphy_1510YP_001558623.1 60 phytofermentans Clostridium xylanase 10 Cphy_0624YP_001557750.1 61 phytofermentans Clostridium xylanase 11 Cphy_2105 XynAYP_001559210.1 62 phytofermentans Clostridium xylanase 10 Cphy_2108YP_001559213.1 63 phytofermentans Clostridium xylanase/  8 Cphy_3207 YYP_001560300.1 64 phytofermentans endoglucanase Clostridium Xylosidase/43 Cphy_0191 YP_001557317.1 65 phytofermentans ArabinofuranosidaseClostridium Xylosidase/ 43 Cphy_0875 YP_001558000.1 66 phytofermentansArabinofuranosidase Clostridium Arabinofuranosidase Cphy_1169YP_001558286.1 67 phytofermentans Clostridium Mannanase 26 Cphy_1071YP_001558190.1 68 phytofermentans Clostridium Mannosidase 26 Cphy_2128YP_001559233.1 69 phytofermentans Clostridium Mannosidase 26 Cphy_2276YP_001559376.1 70 phytofermentans Clostridium Galactosidase Cphy_1936YP_001559043.1 71 phytofermentans Clostridium Endo  5 cel5I AAL79562.172 cellulolyticum Clostridium Exo/Endo 48 CelCCF (dockerin) AAB41452.173 cellulolyticum Cel48F-yeast CO template pMU914 Clostridium Xylosidase39 Ccel_1259 YP_002505595 74 cellulolyticum Clostridium Endo  9Ccel_2226 YP_002506548.1 75 cellulolyticum Clostridium Endo/Exo  9Ccel_0732 YP_002505091.1 76 cellulolyticum (dockerin) Cel9E- yeast COtemplate pMU913 Clostridium Endo  5 Ccel_1099 YP_002505438.1 77cellulolyticum (dockerin) Cel5A- yeast CO template pMU967 ClostridiumEndo/Exo  9 Ccel_2392 YP_002506705.1 78 cellulolyticum (dockerin)Clostridium Endo  9 Ccel_0731 YP_002505090.1 79 cellulolyticum(dockerin) Cel9G- yeast CO template pMU892 Clostridium Endo/Exo  5Ccel_0840 YP_002505196.1 80 cellulolyticum (dockerin) Cel5D- yeast COtemplate pMU891 Clostridium Endo/Exo  8 CelCCC (dockerin) AAA73867.1 81cellulolyticum Cel8C-yeast CO template pMU969 THERMOPHILES AerobesThermobifida fusca xylanase 10 endo-1,4-beta ABL73883.1 82 xylanase(Umxyn10A) Thermobifida fusca xylanase 11 endo-1,4-beta-D- AAV64879.1 83xylanase (xyl11) Thermobifida fusca endo  6 Endoglucanase AAZ55112.1 84Thermobifida fusca exo/endo?  5 Cellulase AAZ56745.1 85 Thermobifidafusca beta-glucosidase  3 exo-1,4-beta- AAZ55642.1 86 glucosidaseThermobifida fusca beta-glucosidase  1 beta-glucosidase AAZ55664.1 87Thermobifida fusca exo/endo 48 cellulose 1,4-beta- YP_290015.1 88cellobiosidase Thermobifida fusca synergy with CBD E8 AAZ55700.1 89endo/exo Thermobifida fusca exo  6 celC (E3) YP_288681.1 90 Thermobifidafusca endo  5 celE (E5) YP_288962.1 91 Thermobifida fusca endo  5 cel5BAAP56348.1 92 (Endoglucanase) Thermobifida fusca endo  9 celA (E1)AAC06387.1 93 Thermobifida fusca endo  6 celB (E2) YP_289135.1 94Thermobifida fusca endo/exo?  9 Tfu_1627 (1,4-beta- YP_289685.1 95cellobiosidase) Anaerobes Clostridium Endo  8 celA (dockerin)YP_001036701.1 96 thermocellum Clostridium Endo/Exo 48 celY (cel48Y)CAI06105.1 97 thermocellum Clostridium Endo  9 Cthe_0625 YP_001037053.198 thermocellum (dockerin) Clostridium Endo  5 celC CAC27410.1 99thermocellum Clostridium Endo  5 Cthe_1471 YP_001037893.1 100thermocellum Clostridium xylanase 10 Cthe_2119 YP_001038519.1 101thermocellum Clostridium beta-glucosidase  1 bglA CAA42814.1 102thermocellum Clostridium beta-glucosidase  3 bglB CAA33665.1 103thermocellum Clostridium arabinofuranosidase 51 Cthe_2548 YP_001038942.1104 thermocellum Clostridium arabinofuranosidase 54 Cthe_1273YP_001037698.1 105 thermocellum Clostridium Endo/Exo  9 Cthe_0040(Cel9I) YP_001036474.1 106 thermocellum Clostridium Endo/Exo  9Cthe_0412 YP_001036843.1 107 thermocellum (dockerin) ClostridiumEndo/Exo  9 Cthe_0825 YP_001037253.1 108 thermocellum (dockerin)Clostridium Endo-xylanase 11 xynA CAD48307 109 stercorarium ClostridiumEndo-xylanase 10 xynB (CelW- CAD48313 110 stercorarium celloxylanase)Clostridium Endo-xylanase 10 xynC (CelX- CAD48314 111 stercorariumcelloxylanase) Clostridium Xylosidase  3 bxlB (b-Xylosidase AJ508405 112stercorarium B) Clostridium Xylosidase 39 bxlA (b-Xylosidase AJ508404113 stercorarium A) Clostridium Xylosidase/beta-  3 bglZ (beta- CAB08072114 stercorarium glucosidase glucosidase) Clostridiumarabinofuranosidase 43 arfA (alpha- AJ508406 115 stercorariumarabinofuranosidase A) Clostridium arabinofuranosidase 51 arfB (alpha-AAC28125 116 stercorarium arabinofuranosidase B) Clostridium Endo  9celZ (Cs-Cel9Z- CAA39010 117 stercorarium Avicellase I) Clostridium Exo48 celY (Cs-Cel48Y- CAA93280 118 stercorarium Avicellase II)Anaerocellum Endo (Exo?) 48 celA (1,4-beta- CAB06786 119 thermophilumglucanase) Anaerocellum Endo  5 celD (EG) CAB01405 120 thermophilumAnaerocellum Endo-xylanase 10 xynA (1,4-beta-D- CAA93627 121thermophilum xylan xylanhydrolase) Anaerocellum Endo  5 celB (EG5)Z86104 122 thermophilum Anaerocellum Endo?  5 Athe_1866 (endo-YP_002573059 123 thermophilum 1,4-beta- mannosidase) Anaerocellum Endo? 5 Athe_0594 YP_002572493 124 thermophilum (“cellulase”) Thermobifidafusca endo/exo  9 Cel9A, TfCel9A- 125 yeast CO gene from restrictiondigest of pMU1248

Example 7: Screening Bacterial Endoglucanases for Expression/Activity inYeast

All of the bacterial endoglucanases were pre-screened for secretedactivity on CMC (FIG. 22). Fifty seven yeast strains expressingbacterial endoglucanases were screened. For each enzyme two differenttransformation clones were assayed. The strains were patched on YPD+Zeoplates (Zeo 250 mg/L) for 2 days and inoculated in 600 uL YPD in 96 wellplates. The strains were grown for 3 days at 35 C at 900 rpm, and theCMC assay (see above) was performed on the supernatants. NegCont isM0749 transformed with empty expression vector pMU1575. TrEG2 in pMU1575was used as positive control construct.

FIG. 22 demonstrates that 15 bacterial enzymes (26%) displayed secretedactivity on CMC. Bacillus subtilis Eg1S and Clostridium cellulolyticumCel5A had secreted activity on CMC similar to the well expressedcontrol, which was T. reesei EG2. The enzymes that demonstrated activityon CMC are listed in the Table 8 below. All genes except BC77, BC80 andBC81 are not codon optimized for yeast; therefore the expression levelof the best genes could be increased further by codon optimization.

Example 8: Synergy of Bacterial Endoglucanases with Yeast Made CBHs onPHW

In order to determine which bacterial endoglucanase increase pretreatedlignocellulose conversion by CBHs, the PHW assay was performed withseveral yeast made bacterial EGs selected by screening on CMC in thepresence of yeast made purified CBH1 and CBH2 (FIG. 23). The assay wasalso supplemented with Novozyme-188 BGL.

FIG. 23 demonstrates that almost all tested bacterial EGs significantlyincrease glucose release from PHW. Additive effect of bacterial EGs wassimilar or higher compared to the positive control—Trichoderma reeseiEG2. Thermobifida fusca celE was particularly successful among the EGs.

Previous work had demonstrated that the T. fusca Cel9A gene is wellexpressed in yeast. We have generated a yeast codon optimized version ofthis gene and expressed it and the native sequence under control of thestrong ENO1 promoter. This resulted in activity on avicel that wasroughly equivalent to that measured for CBH1 candidates (8% conversionin 48 hours, with only Novozymes 188 present as a background). Thisindicated that both the native and the codon optimized version of thegene were well expressed. Thus, this candidate enzyme was tested forsynergy with yeast made, purified CBHs, and T. reesei EG2 in a PHW assay(FIG. 24). As can be seen below, combinations of Cel9A with EG2 havesignificant synergy, and perform better than the individual enzymesadded alone, even though they are twice the concentration.

TABLE 8 List of bacterial endoglucanases demonstrated functionalexpression in yeast (see FIG. 22). BC# Donor organism GHF Gene or locustaq 4 Streptomyces avermitilis 12 endo-1,4-beta-glucanase celA1 34Saccharophagus degradans 5 cel5A 48 Bacillus subtilisendo-1,4-beta-glucanase eglS 56 Clostridium phytofermentans 5 Cphy_3202celulase B 72 Clostridium cellulolyticum 5 cel5I 77 Clostridiumcellulolyticum 5 Ccel_1099 (yeast CO) 80 Clostridium cellulolyticum 5Ccel_0840 (yeast CO) 81 Clostridium cellulolyticum 8 CelCCC (yeast CO)91 Thermobifida fusca 5 celE (E5) 93 Thermobifida fusca 9 celA (E1) 94Thermobifida fusca 6 celB (E2) 95 Thermobifida fusca 9 Tfu_1627 96Clostridium thermocellum 8 celA 99 Clostridium thermocellum 5 celC 108Clostridium thermocellum 9 Cthe_0825 125 Thermobifida fusca 9 Cel9A

Example 9: Characterizing Bacterial Xylanases for Expression/Activity inYeast

Screening was carried out for bacterial genes annotated as xylanasesusing birchwood xylan as the substrate—see protocol above (FIG. 25).Twenty five yeast strains expressing bacterial xylanases were screened.For each enzyme two different transformation clones were assayed. Thestrains were grown in the same manner as the endoglucanases describedabove. All strains have M0749 yeast background. “NegCont” is M0749transformed with empty expression vector pMU1575, and the Trichodermareesei Xyn2 gene cloned into in pMU1575 was used as positive controlconstruct.

FIG. 25 demonstrates that 8 bacterial enzymes (32%) had secretedactivity on xylan. Several xylanases including Clostridiumphytofermentans Cphy1510 (GHF10) and Thermobifida fusca xyl11 hadsecreted activity on xylan similar to or higher than T. reesei Xyn2. Theenzymes that demonstrated activity on xylan are listed in Table 9 below.

TABLE 9 List of bacterial xylanases demonstrated functional expressionin yeast (see FIG. 25). BC# Donor organism GHF Gene or locus taq 13Streptomyces avermitilis 43 endo-1,4-beta-xylanase xynD 51 Bacillussubtilis 11 endo-1,4-beta-xylanase xynA 60 Clostridium phytofermentans10 Cphy_1510 61 Clostridium phytofermentans 10 Cphy_0624 83 Thermobifidafusca 11 endo-1,4-beta-D-xylanase (xyl11) 109 Clostridium stercorarium11 xynA 110 Clostridium stercorarium 10 xynB (CelW-celloxylanase) 111Clostridium stercorarium 10 xynC (CelX-celloxylanase)

Example 10: Synergy of Bacterial Xylanases with Yeast Made CBHs and EG

In order to test synergy of yeast made enzymes with bacterial xylanases,a PHW assay was performed with several yeast made bacterial xylanasespreviously selected by screening on xylan in the presence of yeast madepurified CBH1, CBH2, TrEG2, and yeast made GH43 xylosidase (fromPyrenophora tritici-repentis) (FIG. 26). Trichoderma reesei Xyn2 wasused as the positive control, and a strain expressing an empty vectorserved as a negative control. The assay was also supplemented with ABBGL.

FIG. 25 demonstrates that some bacterial xylanases significantlyincrease glucose release from PHW, especially when external enzyme isnot present. Clostridium phytofermentans GH10 xylanases (BC 60, andBC61) and Clostridium stercorarium XynB (BC110) had the most significanteffect on glucose release from PHW. There are several possibleexplanations for the fact that these xylanases help release glucose. Itis possible that some xylanases also possess endoglucanase or otherhydrolase activity, and thus hydrolyze cellulose directly. Additionally,it is possible that digestion of xylan in the PHW may make the cellulosemore accessible for the cellulases present in the reaction. Increasedrelease of xylose was not measured in the reaction, likely due to thelack of appropriate complementary activities (xylosidase and/oracetylxylanesterase).

Example 11: Cloning and Screening Thermoanaerobacter saccharolyticumXylanases

T. saccharolyticum xylanases were cloned from genomic DNA and fused tothe Enol promoter for expression in S. cerevisiae. A total of 12xylanase-related genes were cloned into the pMU1575 backbone (Table 4).The strains were screened for both xylanase and xylosidase activitiesusing the birchwood xylanase assay and the pNPX xylosidase assay,respectively (FIG. 26). M1594 was the only strain that demonstratedsignificant xylanase activity. No xylosidase activity was detected fromthese strains.

TABLE 10 Description of T. saccharolyticum xylanases cloned andexpressed in yeast. Sample Contig Gene SP Gene Annotation GH Vector #TsX1 Contig 7 or0901 Trans endo-1,4-beta-xylanase precursor 11 pMU1988TsX2 Contig 12 or1447 No Xylan 1,4-beta-xylosidase 39 pMU1989 TsX3Contig 12 or1446 No Xylan 1,4-beta-xylosidase. 52 pMU1990 TsX4 Contig 12or1454 Trans Cellulose 1,4-beta-cellobiosidase- 10 pMU1991 Beta-14-xylanase xynA TsX5 Contig 12 or1455 No Glycosylhydrolase family 10 10pMU1992 TsX6 Contig 12 or1186 SP xylanase/chitin deacetylase pMU1993TsX7 Contig 0 or0277 No xylulokinase pMU1994 TsX8 Contig 0 or0278 Noxylose isomerase xylA pMU1995 TsX9 Contig 0 or0277 No xylulokinase-No SPpMU1996 TsX10 Contig 0 or0278 No xylose isomerase xylA-No SP pMU1997

Example 12: Screening of Bacterial Genes with Mannanase Activity

In order to find an easy, high-throughput screen for cellulases,mannanases, and xylanases, 4 Azurine-Crosslinked Polysaccharides (AZCL)from Megazymes were tested in an agar plate assay. In this assay theenzyme hydrolyzes the insoluble polysaccharide, releasing the solubledye-labeled fragments to provide a “zone of dyeing”. Galactomannan,debranched arabinan, and xylan AZCL attached substrates were tested bythe plate assay. Clones with putative xylanase, and mannanase activityprovided colored zones; however, no arabinase activity was detected onthe debranched arabinan.

Xylanases that demonstrated activity by this plate assay matched theones that were active in previously applied birchwood xylan assay (seeabove). Three functionally secreted yeast made bacterial mannanases(BC68, BC69, and BC70 from C. phytofermentens) were discovered by themannanase plate assay.

Bacterial accessory enzymes expressed by yeast were also screened forsynergy with yeast made enzymes (CBH1, CBH2, EG2, BGL, xylanase,xylosidase) by PHW assay without any external enzymes added (FIG. 28).One enzyme—Clostridium phytofermentans mannosidase (Cphy_2276, GH26,BC70), has a noticeable effect on glucose release from PHW. None of theenzymes had significant effect on xylose release. It is possible thatother activities may be needed in a system in order to notice the effectof some accessory enzymes.

TABLE 11 Summary of functional, “best in class” components expressed inyeast. Hardwood and Paper sludge Additional for Paper sludge Cazyfamily/ Well-Expressed Type of Activity enzyme type Candidates AccessionNumber exoglucanase GH7A (CBH1) T. emersonii See underlined orf CBH1 +HgCBD in pMU1392 GH6A (CBH2) C. lucknowense See omnibus patent CBH2application endoglucanase GH7B (EG1) A. fumigatus EG1 XP_747897 GH5A(EG2) T. reesei EG2 See omnibus patent application GH12A (EG3) N.fischeri EG3 XP_001261563 GH61A (EG4) T. terrestris EG4 ACE10231 GH45A(EG5) C. lucknowense ACH15008 EG5 GH6 (EG6) N. crassa EG6 XP_957415 GH5(bact.) C. cellulolyticum YP_002505438.1 Cel5A GH? (bact.) B. subtilisEGLS CAB13696.2 GH9 (bact.) T. fusca Cel9A AAC06387.1 GH8 (bact.) C.cellulolyticum AAA73867.1 Cel8c xyloglucanase GH74A (EGL6) A. niger XGAAK77227 β-glucosidase BGLI S. fibuligera BGLI See Omnibus patentapplication xylanase GH11 (XYN2) T. reesei xyn2 ABK59833 GH10 A. nigerxyn10 CAA03655.1 β-xylosidase GH3 A. niger Xld3 XP_001389416 GH43 (BXL1)Pyrenophora XP_001940956 tritici-repentis BXL beta-mannase GH5 (MAN1) A.aculeatus MAN5 AAA67426 beta- GH2/GH26 C. phytofermentens mannosidasemannosidase acetylxylanesterase CE1 (AXE) N. fischerii AXE1 XP_001262186arabinofuranosidase GH54 (ABF1) A. niger ABFB AAA93264 ferulic CE1(FAEA) A. niger FAEA XP_001393337 acid/cinnamoyl CE1 (FAEB) T.stipitatus FAEB EED17739 esterase A-glucuronidase GH67 Pichia stipitisABN67901 glucuronyl CIP2 C. globosum XP_001226041 esterase

Example 13: Combinations of Components to Enhance Hydrolysis: Effect ofDifferent EG Combinations (Pair Wise Combinations) on PHW Conversion byYeast Made CBHs in the Presence of External Enzymes (EE)

In order to determine if different EGs were synergistic with each other,PHW assays were used to analyze EG combinations with the goal ofdetermining synergistic relationships. If the EGs had similar functions,then combinations of the EGs should be no better than a single EG(either) loaded at twice the concentration. However, if the EGs weresynergistic, then combinations should yield greater hydrolysis than a 2×concentration of either enzyme.

To test pair wise combinations, a PHW assay was performed with thesupernatants of yeast strains expressing individual EGs (Table 4)combined in pairs (1 ml+1 ml) in all possible combinations. The EGexpressing strains were patched on YPD+Zeo plates for 1 day (exceptM1023 that was patched on SD-URA), inoculated in YPD in shake flasks andgrown for 72 hours. The strain expressing an empty vector was used asnegative control (2 ml, NC). The strains expressing single EGs (2 ml or1 ml+1 mlNC) were used as positive controls. All samples including NCwere supplemented with 1 mg/g CBH1, 1 mg/g CBH2, and 1 mg/g AB BGL (FIG.29) or 1 mg/g CBH1, 1 mg/gCBH2, 0.2 mg/g BGL, and 1 mg/g Zoomerase (FIG.29).

TABLE 12 Yeast strains expressing EGs of different GH families GHFStrain Organism Donor Gene Host GH7 M1311 Fungi Aspergillus fumigatusEG1 M0509 GH5 M1450 Fungi Trichoderma reesei EG2 M0749 GH12 M1378 FungiNeosartorya fischeri EG3 M0509 GH61 M1391 Fungi Chaetomium globosum EG4M0509 GH45 M1420 Fungi Chaetomium globosum EG5 M0544 GH6 M1400 FungiNeurospera crassa EG6 M0509 GH8 M1456 Bacteria Clostridiumcellulolyticum Cel8C(BC81) M0749 GH9 M1023 Bacteria Thermobifida fuscaCel9A(BC125) M0749 GHX M1454 Bacteria Bacillus subtilis EglS(BC48) M0749

All EGs expressed on 2 u plasmid under ENO pr/tt containing URA3 and Zeomarkers. Backbone vector pMU1531 for fungal EGs; pMU1575 for bacterialEGs. Fungal EGs have native signal sequences; bacterial EGs attached toS.c.Invertase signal. Strains with fungal EGs were selected on YPD+Zeoplates; strains with bacterial EGs were selected on SD-URA—plates.

As can be seen from FIG. 29, several combinations of EGs outperformed a2× loading of either enzyme, indicating that the EGs are indeedsynergistic. Even though there was some overlap in synergy betweendifferent time-points (27 and 48 hrs), the amount of synergy waschanging over time.

In order to analyze the EG pairs experiment data the Tables 13A and 13Bwere composed based on FIGS. 31 and 32 data. In these Tables twoparameters were calculated for each EG pair: activity (bold fontnumbers)—increase in glucose release compared to NegCont; and synergy(normal font numbers)—increase in glucose release compared to the moreactive component of the couple. Activity was calculated by deducting theglucose release value for negative control from glucose release valuefor EG couple. Synergy was calculated as % of increase in glucoserelease for EG pair compared to the glucose release for the more activecomponent of the pair. The data presented on FIGS. 33 and 34 and Tables13A and B demonstrated that:

1. EG combinations have a definite advantage in PHW cellulose conversioncompared to single EGs.

2. In early PHW conversion time points each of the 9 EGs (from separatefamilies) are synergistic with some other EG.

3. The synergy effect becomes less noticeable at the later time ofconversion.

In order to select the most efficient EG couples, the best EG pairs wereranged based on both parameters: activity and synergy, for bothtime-points (Table 14). The pairs in bold font in the Table 14 arepresent in all four “winning” groups and considered as the mostefficient EG combinations for these experimental conditions.

TABLE 13 Data analysis of experiment with different EG combinations. PHWglucose 27 h PHW glucose 48 h GH 5 7 12 61 45 6 8 9 x GH 5 7 12 61 45 68 9 x 5 0.38 0 0 0 0 0 0 28 21 5 0.83 4 7 0 0 4 0 9 0 7 0.39 0.35 17 7 00 0 15 16 7 1.00 0.48 5 6 8 7 5 11 0 12 0.44 0.79 0.32 12 13 0 0 10 0 121.09 0.92 0.76 8 3 6 0 12 0 61 0.36 0.53 0.65 0.20 ? ? 0 15 10 61 0.910.94 1.05 0.73 ? ? ? 5 0 45 0.30 0.45 0.65 ? 0 7 14 0 8 45 0.55 0.760.88 ? 0.40 0 0 0 0 6 0.28 0.46 0.35 ? 0.21 0 18 0 6 6 1.00 0.79 1.00 ?0.41 0.54 0 0 0 8 0.18 0.31 0.37 0.10 0.28 0.47 0 6 17 8 0.65 0.64 0.82? 0.35 0.49 0.46 0 0 9 1.09 0.82 0.67 0.82 0.44 0.35 0.57 0.41 5 9 1.271.35 1.40 1.13 0.76 0.81 0.96 0.95 0 x 0.95 0.78 0.41 0.46 0.19 0.180.40 0.55 0 x 0.48 0.45 0.57 0.36 0.17 0.33 0.20 0.70 0 Bold fontnumbers denote activity - increase in glucose release compared toNegCont (CBHs + EE), g/l (EG couple activity minus NC); normal fontnumbers denote - Synergy - increase in glucose release compared to themore active component of the couple, % (100%*EG couple act. divided byEG max act. minus 100%). A - 27 h time-point; B - 48 h time-point.

Bold font numbers denote activity—increase in glucose release comparedto NegCont (CBHs+EE), g/l (EG couple activity minus NC): normal fontnumbers denote—Synergy—increase in glucose release compared to the moreactive component of the couple, % (100%*EG couple act. divided by EG maxact. minus 100%). A—27h time-point; B—48h time-point.

TABLE 14 Data analysis of experiment with different EG combinations (seeFIGS. 28 and Table 13). Activity Synergy 27 hrs 48 hrs 27 hrs 48 hrsRang EG couple Activity EG couple Activity EG couple Synergy EG coupleSynergy # GH/GH g/l GH/GH g/l GH/GH % GH/GH % 1  5/9 1.09  9/12 1.40 5/9 28  9/12 12 2  5/X 0.95 9/7 1.35  5/X 21 9/7 11 3  9/7 0.82 9/51.27  6/8 18 9/5  9 4  9/61 0.82  9/61 1.13  7/12 17  7/45  8 5  7/120.79  5/12 1.09  X/8 17 61/12  8 6  7/X 0.78 61/12 1.05  7/X 16  5/12  77  9/12 0.67  6/12 1.00  9/7 15 7/6  7 8 61/12 0.65 6/5 1.00  9/61 15 7/61  6 9 12/45 0.65 7/5 1.00 45/8 14 12/6   6 10 12/45 13  7/12  5 1161/12 12 7/8  5 12  9/12 10  9/61  5

In this Table the best performing EG pairs based on activity and synergydata from Table 13 listed in the order of performance starting with thebest pairs. Four groups of the best performers were formed for twodifferent parameters (activity and synergy) and for two differenttime-points: 27 and 48 hrs. The pairs bold font are the pairs thatpresent in all 4 groups.

Example 14: Testing of Higher EG Combinations for Enhanced PHW Activity

Based on the EG pairs screening above, an experiment was designed inwhich the most efficient EG pairs were combined with each of theremaining EGs from Table 14. The PHW assay was performed with allpossible triple EG combinations at the presence of external enzymes (EE,see composition above) and yeast made CBHs (FIG. 30). The total assayvolume was divided into 3 parts for the triples (0.67 mL each), whereasit was divided into only 2 or 1 part for the pair and single controls,respectively.

FIG. 30 demonstrates that yeast cellulytic system, when used with EE,does benefit from more complex EG compositions. Based on 48 hrs data twobest EG triplets were selected for further experiments: GH9+GH5+GH12 andGH9+GH5+GH7.

The best triplets were combined with each of the remaining EGs and thePHW assay was repeated again at two different concentrations of EE (FIG.31). FIG. 31 demonstrates that:

1. EG combinations have a definite advantage in PHW glycan conversioncompared to single EGs.

2. Which EG combination is the best depends on the EE load and time ofconversion.

3. At all times and EE loads tested the best EG combos include:Cel9A(GH9), EG3(GH12), EG1(GH7) and EG2(GH5).

4. At lower EE loadings, the combination of GH5, GH9, GH7, and GH12appears the best.

The data for the best single EGs (GH9 and GH5) and the best four EGstogether (GH9, GH5, GH12, GH7) were plotted as a time course of PHWconversion at the presence 2 mg/g EE next to the controls—2 mg/g and 4mg/g EE without EGs added (supernatant of empty vector added instead)(FIG. 32). FIG. 32 demonstrates that the four EG combination has adefinite advantage over the best single EGs at the same volume. Also,FIG. 32 demonstrates that the best EG combination provides increase inPHW conversion equivalent to 2 mg/g EE.

Example 15: Expression of a “Complete” System of Enzymatic Components toDigest Lignocellulose

The technical challenge of developing a “complete” or mostly completelignocellulolytic enzyme system for expression in yeast, is that thissystem is likely to consist of many components. These components willneed to be expressed in multiple copies in order to generate enoughactivity to be meaningful. Thus, developing tools for multi-gene,multicopy expression are very useful in this context.

Transferable System for Expressing Multiple Genes in Multiple Copies

Expressing multiple copies of the ˜25 gene types listed in Table 4, inaddition to the “core” enzymes (CBH1, CBH2, EG2, and BGL) alreadyproduced in yeast, will require new molecular tools. Repeatedintegration with marker removal will be labor intensive. In addition tothis, a system that would make the enzyme system transferable betweenstrains would extremely valuable since new hosts are continually beingcreated.

Expressing large pieces of DNA is a solution to the problem outlinedabove. Among the options for expressing large pieces of DNA are CENbased plasmids and Yeast artificial chromosomes (YACs). “CEN” refers tocentromeric, and CEN elements allow high fidelity dispersion of geneticelements into mother and daughter cells during cell division. Firstdeveloped in 1987 (Burke D T, Carle G F, Olson M V, “,” Science. 1987May 15; 236(4803):806-12), YACs have been used for cloning very largepieces of DNA for expression in non-yeast hosts (e.g. in mice; Schedl,1993), and for genome sequencing (e.g. Krzywinski M, Wallis J, Gosele C,et al., “Integrated and sequence-ordered BAC- and YAC-based physicalmaps for the rat genome,” Genome Res. 2004 April; 14(4):766-79). Theyare able to maintain up to 3 megabases of DNA. Of particular interestfor our project, YACs have been developed whose copy number can beamplified (Smith D R, Smyth A P, Moir D T., “Amplification of largeartificial chromosomes,” Proc Natl Acad Sci USA. 1990 November;87(21):8242-6). This is based on disrupting CEN function, and selectingfor cells with asymmetric segregation of the YAC. The authors showedthat the system developed could increase the copy number of a 560 Kb YACto 13 copies, and of 120 Kb YAC to 20 copies. After 20 generations the560 Kb YAC had fallen to 8.2 copies, and the 120 Kb YAC had fallen to11.3 copies. These results indicate that even these very large DNAfragments, with no, or little selective benefit to the cell can bemaintained with decent stability. The copy number feature for YACs wasoriginally created in CEN plasmids (Chlebowicz-Sledziewska E,Sledziewski A Z., “Construction of multicopy yeast plasmids withregulated centromere function,” Gene. 1985; 39(1):25-31), and theseplasmids are likely the easiest option for expressing the ˜20 kb pieceof DNA that would comprise the “major” activities. In addition to thesefeatures, researchers (Spencer F, Simchen G. “Transfer of YAC clones tonew yeast hosts,” Methods Mol Biol. 1996; 54:239-52) have shown thatYACs can be transferred from one yeast host to another, as well as beingmodified by homologous recombination.

For enzymes that are deemed necessary in only a single, or doublecopy—“minor” components—a single large integrative construct can bebuilt, which will save the effort of producing a large CEN plasmid, andcreate a more stable system.

Example 16: Assembly of Large Vectors for Expression of Multiple Genes

Assembly of genes into large constructs by homologous recombination iswell known in S. cerevisiae (Shao Z, Zhao H, Zhao H., “DNA assembler, anin vivo genetic method for rapid construction of biochemical pathways,”Nucleic Acids Res. 2009 February; 37(2):e16. Epub 2008 Dec.12)(Oldenburg K R, Vo K T, Michaelis S, Paddon C.,“Recombination-mediated PCR-directed plasmid construction in vivo inyeast,” Nucleic Acids Res. 1997 Jan. 15; 25(2):451-2). This represents atool for both routine cloning and for combining many genetic elements atonce. Using the enzymes tested above, we were able to assemble large CENconstructs for expression of multiple genes in multiple copies. Thesevectors were constructed with one of two markers (hph or zeocin marker),with the ARS1 origin of replication from S. cerevisiae, with adisruptable centromere (CEN 4), and with a 2 micron element present.This disruptable element was made by placing the inducible Gall promoterupstream of the centromere. During growth on galactose, the plasmidbecomes unstable.

FIG. 33 demonstrates the ability to assemble four endoglucanasessimultaneously into a single vector (EG1 from A. fumigatus under thecontrol of the ENO1 promoter/PYK terminator, EG4 from C. globosum underthe control of the FBA promoter/PGI terminator, EG5 from C. lucknowenseunder control of the GPM1 promoter and TPI terminator, and EG6 from C.globosum under control of the ENO2 promoter and TDH3 terminator). Eachcassette for expression was amplified by PCR with overlapping sequencesthat could recombine to form the final vector shown (actual vector iscircular, not linear). Several colonies picked from this transformationall had activity on CMC, indicating that the EGs were functionallyexpressed. The construct (pMU1943) was verified by carrying out PCRacross all of the junctions of the individual pieces that wereassembled. The yeast strain containing this cassette was called M1509.

As outlined above, a similar CEN vector and strain were created with thezeocin marker (pMU1666). EG1, EG4, EG5 and EG6 were successfullyassembled by YML into CEN vectors with the zeocin marker (strain M1553).PCR tests were done to confirm the junctions between EG cassettes andbetween vector and cassettes for the first (EG1) and last (EG4)cassettes.

CEN vectors were also built that had either 7 genes or 11 genes viayeast mediated ligation. Schematics for these two vectors are shown inFIG. 34. These vectors were tested to verify the presence of the insertsvia PCR. The two vectors below demonstrate that vectors as large as 23kB and 35 kB, respectively can be generated in this manner.

Example 17: Amplification of CEN Vectors for Multicopy Expression

Strain M1509 produced very few slow-growing colonies at TO at ahygromycin concentration of 1000 μg/ml. After growth in YP+galactose,there was an increased number of colonies on hygromcin 1000. Thesecolonies also grew faster on YPD+hygromycin 1000 than colonies beforethe galactose treatment. This suggested that the copy number may haveincreased with the galactose treatment allowing faster growth and morecolonies on the high hygromycin concentration plate. However, a CMCassay revealed that the endoglucanase activity both before and after thegalactose treatment remained almost the same (FIG. 35).

Outgrowth was also done in YPD without antibiotic for about 10generations and the CMC activity before and after the outgrowth remainedfairly similar indicating the stability of the plasmid (FIG. 36).Another interesting feature was that colonies from YPD plate (noselection) after a galactose growth treatment showed variable CMCactivity, with some colonies having a large decrease in activity(indicated by a very high standard deviations in FIG. 35). Thisindicates that the CEN vector was working as expected in presence ofgalactose causing some cells to retain more copies of plasmid and othersto lose it.

As noted above, M1553 is a strain containing a CEN vector with thezeocin resistance cassette and four endoglucanases EG1, EG4, EG5 andEG6. This strain was tested for antibiotic resistance and EG activity.Initially M1553 could grow up to a zeocin concentration of 50 μg/ml inYPD plates, and this strain passaged in YPG (galactose) and zeocin at 50μg/ml showed colonies when plated on YPD plates with zeocin at 100 μg/m.These zeocin (100)-resistant colonies also grew on YPD-zeo 500 ug/mLplates when re-streaked. Ten colonies from the YPD-zeo 100 ug/mL platewere compared against ten original CEN strain colonies grown on YPD—zeo50 ug/mL. Serial dilutions 1:5, 1:10, 1:20 and 1:40 were made fromculture supernatants and a CMC assay was carried out on the dilutedsupernatants.

FIG. 37 shows a comparison of the average performance of the top 3colonies from each of these plates at the different dilutions. Coloniesfrom the 100 ug/mL zeocin plate perform better than the zeocin 50 ug/mLcolonies indicating that amplification of the CEN vector has occurred.Depending on the dilution analyzed (the CMC assay appears to be atsaturation in some dilutions), a 1.5 to 2× difference in CMCase activitycan be observed between the two sets of top colonies.

This demonstrates that growth in galactose to disrupt CEN functioncoupled with selection via the zeocin marker can result in vectoramplification.

Example 18: Activity of a CEN Vector with Multiple EGs on PHW

A CEN vector with the zeocin resistance marker expressing the A.fumigatus EG1, C. globosum EG4, C. lucknowense EG5, and C. globosum EG6from different promoters and terminators was created in M0544 asdescribed above. This vector was tested for its effect on PHW hydrolysisin an unamplified state along with strains expressing EG3 and Cel9A from2 micron vectors (FIG. 38). The results indicate that a 2× loading of astrain producing high levels of the core enzymes (M1179) is equivalentto a 1× loading of M1179 plus a 1× loading of the CEN vector strain (orto a 1× loading of M1179 and a mixture of the CEN strain, EG3, andCel9A).

Example 19: Screening of Amylolytic Enzymes for Expression in Yeast

Over one hundred amylolytic, cellulolytic, and accessory enzymes fromyeast, fungi, bacteria and plants were screened for functionalexpression in yeast. Most of the enzymes that were selected forscreening are summarized in Tables 15 and 16. The bacterial enzymesmarked “BC” are described in Table 7. The enzymes from Tables 15 and 16were expressed in yeast and screened by multiple assays individually orin combinations. Table 15 includes 67 genes (first 10 overlap with Table16). For 32 genes functional expression in yeast was confirmed. Table 16contains 81 genes; for 18 genes functional expression in yeast wasconfirmed (rows bolded). The information about gene gene sequences wasobtained from NCBI database or from proprietary Mascoma genomesequencing data (marked * in the Table 16). The genes were eithersynthesized (GeneArt or DNA2.0) or PCR amplified. Synthetic genes wereeither native DNA sequences or codon optimized for S. cerevisiae. WhenPCR was used to obtain genes, either genomic DNA or cDNA was used astemplate. The genes used are described in the Tables 15, 16, or Table 7.The sequences of the important genes used for construction of CBPstrains are listed in Table 19. The genes were expressed under ENO1promoter and terminator from 2-micron plasmid pMU1575 (FIG. 41). Thegenes were inserted between PacI/AscI sites of pMU1575 either by cloningor yeast mediated ligation. Yeast and fungal genes were expressed withtheir native signal sequences. Bacterial genes (such as AE49) wereattached to S. cerevisiae Invertase signal sequence. Expressionconstructs were transformed into an industrial background strain M1744,M509, or M0139 and selected on minimal URA deficient media.Transformants were grown in YPD for 3 days and supernatants wereanalyzed for activity. Data for the most active alpha-amylases (AA),glucoamylases (GA) and alpha-glucosidases (AGL) screened by starch-DNS,starch-GHK, maltose and Corn mash assays are summarized in Table 17. Theexample of screening of several enzymes for functional expression inyeast demonstrated on FIG. 42. Secreted activity of strains expressingsynthetic genes was measured by Starch-DNS, Starch-GHK, and Maltoseassays. FIG. 42 demonstrates that different enzymes have differentactivity on different substrates revealing different mechanisms ofaction.

TABLE 15 Amylolytic and other enzymes that were approved by FDA for feedand/or food use screened for functional expression in yeast. Grey boxesindicate enzymes that demonstrated functional expression in yeast. SE#AE# Organism Source Enzyme Protein ID 1 6 Bacteria Bacillus subtilisAlpha-amylase AAA22194.1 2 13 Bacteria Bacillus subtilis Alpha-amylaseACM91731.1 3 14 Bacteria Bacillus subtilis Alpha-amylase CAL64397.1 4 17Bacteria Bacillus subtilis Maltogenic AAF23874.1 alpha-amylase 5 15Bacteria Bacillus subtilis Pullulanase AAC00283.1 6 16 Bacteria Bacillussubtilis Isomaltase? AAG23399.1 7 18 Bacteria Bacillus subtilisIsomaltase? BAA23408.1 8 19 Bacteria Bacillus subtilis Isomaltase?ZP_03592917.1 9 20 Bacteria Bacillus subtilis Isomaltase? BAA22245.1 102 Yeast Saccharomyces Glucoamylase AAA35107.1 cerevisiae 11 FungiAspergillus Glucoamylase AAP04499.1 niger 12 Fungi AspergillusGlucoamylase BAA01540.1 oryzae 13 Fungi Rhizopus Glucoamylase BAA00033.1oryzae 14 Fungi Aspergillus Alpha- BAA23616.1 niger glucosidase 15Bacteria Bacillus Alpha-amylase CAA01355.1 licheniformis 16 BacteriaBacillus Pullulanase AAU24646.1 licheniformis 17 Bacteria BacillusPullulanase ABE68909.1 acidopullulyticus 18 Bacteria Bacillus subtilisProtease ABJ99976.1 19 Bacteria Bacillus Protease AAZ77709.1licheniformis 20 Fungi Aspergillus Beta-glucosidase CAB75696.1 niger 21Fungi Talaromyces CBH1 AAL89553 emersonii 22 Fungi Trichoderma CBH2AAA34210.1 reesei 23 Fungi Trichoderma EG1 AAA34212.1 longibrachiatum 24Fungi Trichoderma EG2 ABA64553.1 reesei 25 Fungi Trichoderma EG3BAA20140.1 reesei 26 Fungi Trichoderma Xylanase CAA49294.1 reesei 27Fungi Aspergillus Xylosidase CAK37179.1 niger 28 Fungi AspergillusXylosidase/Arabinofuranosidase CAK39870.1 niger 29 Fungi AspergillusFerulic acid CAA70510.1 niger esterase 30 Fungi AspergillusAlpha-amylase CAA36967.1 niger 31 Fungi Aspergillus Alpha-amylaseCAA36966.1 niger 32 Fungi Aspergillus Xylanase AAS46914.1 niger 33 FungiAspergillus Xylanase AAS46913.1 niger 34 Fungi Aspergillus XylanaseCAA03655.1 niger 35 Fungi Aspergillus Isopullulanase BAA19473.1 niger 36Fungi Aspergillus Alpha-amylase XP_001402054.1 niger 37 FungiAspergillus Endopolygalacturonase XP_001389562.1 niger 38 FungiAspergillus Pectinase CAK42510.1 niger 39 Fungi AspergillusArabinofuranosidase CAK42333.1 niger 40 Fungi Aspergillus ProteaseXP_001401093.1 niger 41 Plant Zea mays Pullulanase NP_001104920.1 42Plant Oryza sativa Pullulanase ACY56113.1 43 Plant Zea mays IsoamylaseACG43008.1 44 Fungi Aspergillus Lipase ABG73613.1 niger 45 FungiAspergillus Lipase ABG73614.1 niger 46 Bacteria Bacillus XylanaseABF61784.1 licheniformis 47 Fungi Humicola Xylanase CAA53632.1 insolens48 Fungi Talaromyces Xylanase CAD34597.1 emersonii 49 Fungi TrichodermaXylanase AAQ67413.1 viride 50 Plant Triticum Pullulanase ABL84490.1aestivum 51 Yeast Saccharomyces Endopolygalacturonase NP_012687.1cerevisiae 52 Yeast Kluyveromyces Endopolygalacturonase AAR84199.1marxianus 53 Bacteria Bacillus subtilis Pectin lyase NP_389746.1 54Bacteria Bacillus Polygalacturonase YP_080606.1 licheniformis 55Bacteria Bacillus Pectin lyase YP_079258.1 licheniformis 56 FungiAspergillus Endopolygalacturonase CAB72125.1 niger 57 Fungi AspergillusEndopolygalacturonase CAB72126.1 niger 58 Fungi AspergillusEndopolygalacturonase XP_001390812.1 niger 59 Fungi AspergillusEndopolygalacturonase CAB72931.1 niger 60 Fungi AspergillusEndopolygalacturonase CAK44164.1 niger 61 Fungi Aspergillus Pectin lyaseCAK48529.1 niger 62 Fungi Aspergillus Pectin lyase CAK37997.1 niger 63Fungi Aspergillus Pectin lyase AAW03313.1 niger 64 Fungi AspergillusPectin lyase CAK47350.1 niger 65 Fungi Aspergillus Pectin lyaseACE00421.1 niger 66 Fungi Trichoderma Acetyl Xylan Q99034 reeseiEsterase 67 Fungi Aspergillus Feruoyl esterase XP_001393337 niger 60Fungi Aspergillus Endopolygalacturonase CAK44164.1 niger 61 FungiAspergillus Pectin lyase CAK48529.1 niger 62 Fungi Aspergillus Pectinlyase CAK37997.1 niger 63 Fungi Aspergillus Pectin lyase AAW03313.1niger 64 Fungi Aspergillus Pectin lyase CAK47350.1 niger 65 FungiAspergillus Pectin lyase ACE00421.1 niger 66 Fungi Trichoderma AcetylXylan Q99034 reesei Esterase 67 Fungi Aspergillus Feruoyl esteraseXP_001393337 niger

TABLE 16 Amylolytic and other enzymes screened for functional expressionin yeast. AE# Organism Source Enzyme Protein ID 1 Yeast

Alpha-amylase CAA29233.1

2 Yeast Saccharomyces Glucoamylase AAA35107.1 cerevisiae 3 Yeast

Glucoamylase AAA33923.1

4 Yeast

Alpha-glucosidase CAA39501.1

5 Yeast

Alpha-amylase AAB21151.2

6 Bacteria

Alpha-amylase AAA22194.1 7 Yeast

Alpha-amylase CAA51912.1

8 Yeast

Glucoamylase CAA41120.1

9 Yeast

Glucoamylase CAC83969.1

10 Yeast

Alpha-glucosidase CAF31354.1

11 Yeast Lipomyces Alpha-amylase AAC49622.1 kononenkoae (pullulanase?)12 Yeast

Alpha-amylase AAO12212.1

13 Bacteria

Alpha-amylase ACM91731.1 14 Bacteria Bacillus subtilis Alpha-amylaseCAL64397.1 15 Bacteria Bacillus subtilis Pullulanase AAC00283.1 16Bacteria Bacillus subtilis Isomaltase? AAG23399.1 17 Bacteria Bacillussubtilis Maltogenic alpha-amylase AAF23874.1 18 Bacteria Bacillussubtilis Isomaltase? BAA23408.1 19 Bacteria Bacillus subtilisIsomaltase? ZP_03592917.1 20 Bacteria Bacillus subtilis Isomaltase?BAA22245.1 21 Bacteria Clostridium Alpha-amylase ABX42302phytofermentans 22 Bacteria Clostridium Alpha-amylase/pullulanaseABX42665 phytofermentans Type I 23 Bacteria Clostridium PullulanaseABX42692 phytofermentans 24 Bacteria Clostridium Alpha-amylase ABX42702phytofermentans 25 Bacteria Clostridium Alpha-amylase ABX42703phytofermentans 26 Bacteria Clostridium amylo-1,6-glucosidase ABX42704phytofermentans (debranching) 27 Bacteria Clostridium Alpha-amylaseABX42705 phytofermentans 28 Bacteria Clostridium Alpha- ABX42711phytofermentans amylase/neopullulanase 29 Bacteria ClostridiumAlpha-glucosidase ABX44132 phytofermentans 30 Bacteria ClostridiumAlpha-xylosidase ABX40605 phytofermentans 31 Bacteria

Alpha-xylosidase/alpha- ABX42246

glucosidase 32 Bacteria Clostridium Alpha-amylase ABN52030 thermocellum33 Bacteria Clostridium Glucoamylase ABN53008 thermocellum 34 BacteriaClostridium Amylo-alpha-1,6- ABN51356 thermocellum glucosidase(debranching) 35 Bacteria Clostridium Amylo-alpha-1,6- ACL76625cellulolyticum glucosidase (debranching) 36 Bacteria ClostridiumGlucoamylase ACL74721 cellulolyticum 37 Bacteria Thermobifida fuscaAlpha-amylase AAZ54623 38 Bacteria Thermobifida fuscaAlpha-amylase/maltotriose- AAZ55023 producing alpha-amylase 39 BacteriaThermobifida fusca Alpha-glucosidase AAZ54871 40 Bacteria Thermobifidafusca Glucoamylase? AAZ54084 41 Bacteria Thermobifida fuscaGlucoamylase? AAZ55383 42 Bacteria Thermobifida fuscaAlpha-glucosidase/Alpha- AAZ55648 xylosidase 43 Bacteria AnaerocellumAlpha-amylase/pullulanase ACM59580 thermophilum Type I 44 BacteriaAnaerocellum Alpha-amylase/pullulanase ACM59734 thermophilum Type I 45Bacteria Anaerocellum Glucoamylase (GH 15- ACM59378 thermophilumrelated) 46 Bacteria Anaerocellum alpha-xylosidase ACM61134 thermophilum47 Bacteria Thermoanaerobacterium Alpha- * saccharolyticumamylase/amylopullulanase 48 Bacteria Thermoanaerobacterium alpha- *saccharolyticum amylase/cyclomaltodextrinase 49 Bacteria

Glucoamylase *

50 Bacteria Thermoanaerobacterium Glucoamylase * saccharolyticum 51Bacteria Thermoanaerobacterium Amylopullulanase AAA19800 saccharolyticum52 Bacteria Streptomyces Alpha-amylase/oligo-1,6- BAC69017 avermitilisglucosidase 53 Bacteria Streptomyces alpha-glucosidase BAC69435avermitilis 54 Bacteria Streptomyces Isoamylase/glycogen BAC69862avermitilis debranching enzyme 55 Bacteria StreptomycesIsoamylase/glycogen BAC70500 avermitilis debranching enzyme 56 BacteriaStreptomyces alpha-glucosidase BAC73692 avermitilis 57 BacteriaStreptomyces Alpha-amylase BAC73693 avermitilis 58 Bacteria StreptomycesPullulanase BAC73694 avermitilis 59 Bacteria StreptomycesAmylo-alpha-1,6- BAC69169 avermitilis glucosidase (debranching) 60Bacteria Streptomyces Amylo-alpha-1,6- BAC73363 avermitilis glucosidase(debranching) 61 Bacteria Streptomyces Amylo-alpha-1,6- BAC73364avermitilis glucosidase (debranching) 62 Bacteria StreptomycesGlucoamylase (GH 15- NP_827612 avermitilis related) 63 BacteriaStreptomyces Glucoamylase (GH 15- NP_827679 avermitilis related) 64Bacteria Streptomyces Glucoamylase (GH 15- NP_821272 avermitilisrelated) 65 Bacteria Streptomyces Glucoamylase (GH 15- NP_823108avermitilis related) 66 Bacteria Bacillus subtilis ARA1 arabinoxylanaseCAB13699.1 67 Bacteria

ARA2 arabinan endo-1,5- CAB15969.1 alpha-L-arabinosidase 68 BacteriaBacillus subtilis ARA3 arabinan-endo 1,5- CAA99586.1 alpha-L-arabinase69 Bacteria Bacillus subtilis ARA4 arabinan endo-1,5- AL009126alpha-L-arabinosidase 70 Bacteria Bacillus subtilis ARA5 endo-arabinaseD85132 71 Bacteria Clostridium ARA6 Arabinogalactan CP000885phytofermentans endo-1,4-beta-galactosidase 72 Bacteria

ARA7 arabinan-endo 1,5- AAU40201.1

alpha-L-arabinase 73 Bacteria

ARA8 arabinan-endo 1,5- AAU41895.1

alpha-L-arabinase 74 Bacteria Bacillus ARA9 arabinogalactan endo-AAU43089.1 licheniformis 1,4-beta-galactosidase 75 Bacteria BacillusARA10 arabinan endo-1,5- AAU43033.1 licheniformis alpha-L-arabinosidase76 Bacteria Bacillus ARA11 endo-1,4-beta- AAU39947.1 licheniformisxylanase 77 Bacteria Thermoanaerobacterium ARA12 Arabinogalactan *saccharolyticum endo-1,4-beta-galactosidase 78 BacteriaThermoanaerobacterium ARA13 Alpha-N- * saccharolyticumarabinofuranosidase 79 Yeast Arxula Glucoamylase CAA86997.1adeninivorans 80 Bacteria Klebsiella Pullulanase ACI10956.1 pneumoniae81 Fungi Hormoconis Glucoamylase CAA48243.1 resinae 82 FungiAureobasidium Glucoamylase ADN65121.1 pullulans * - the gene sequencewas obtained from genome sequence sequenced by Mascoma.

TABLE 17 Activity screening summary for yeast made alpha-amylases (AA),glucoamylases (GA), and alpha-glucosidases (AGL). Amount of plusesreflects relative activity level. NT - not tested. CO - codon optimized.Strains express individual enzymes on 2u vector pMU1575 in 0509 or M0139background strains. Activity Assay DNS GHK Corn Starch Starch MaltoseMash AE# SE# Source Enzyme AA/GA GA AGL All Strain*  1 —Saccharomycopsis AA ++ − + M1910 fibuligera  5 — Debaryomyces AA ++ − +M1911 occidentalis  6  1 Bacillus subtilis AA ++ + + ++ M1912  7 —Debaryomyces AA ++ − − ++ M1913 occidentalis 11 — Lipomyces AA − − − NT— kononenkoae 12 — Lipomyces AA + + − NT M1914 kononenkoae 13  2Bacillus subtilis AA ++ + + M1915 — 15 Bacillus AA ++ − − ++ M1916licheniformis — 30 Aspergillus niger AA − − − − — Saccharomyces GA − − −− —  2 10 cerevisiae  3 — Debaryomyces GA − + ++ − M1917 occidentalis  8— Saccharomycopsis GA +++ +++ +++ +++ M1918 fibuligera  8CO —Saccharomycopsis GA + + + NT — fibuligera  9 — Saccharomycopsis GA ++++++ +++ NT M1919 fibuligera 49 — T. sacch GA + ++ ++ +++ M1920 — 11Aspergillus niger GA ++ +++ + +++ M1921 — 11CO Aspergillus niger GA − −− − — — 12 Aspergillus GA − − − NT — oryzae — 13 Rhizopus oryzae GA − −− NT —  4 — Pseudozyma AGL − − − + M1922 tsukubaensis 10 —Saccharomycopsis AGL − − +++ NT M1923 fibuligera — 14 Aspergillus nigerAGL − − +++ +++ M1924 *Strains exnressing individual enzymes on 2uvector pMU1575 in M0509 or M0139 backaround strains — — genes PCRed NTNot tested — — genes ordered from GeneArt − No Activity — — genesordered from DNA + Some 2.0 Activity ++ Good Activity +++ Best Activity

Example 20: Screening of Amylolytic and Accessory Enzymes for Synergywith AE8

Particular combinations of hydrolytic enzymes were selected for the bestconversion of particular substrates such as corn mash. This was achieveddue to screening of over one hundred enzymes for functional expressionin yeast, synergy with each other, and performance in industriallyrelevant bioprocess conditions. Particular combinations include: AE9;AE9+AE8; AE9+AE1; AE9+AE7; AE9+AE10; AE9+AE8+AE10; AE9+AE7+AE10;AE9+AE7+AE8+AE10; AE1+AE8+AE9+AE10; and all other combinations of AE1,AE7, AE8, AE9, and AE10 (see Tables 16 and 19). Other particularcombinations of hydrolytic enzymes that demonstrated high glucoserelease from substrates such as pretreated corn fiber and corn syrup(concentrated liquid fraction left after corn mash fermentation)include: “core” cellulases, xylanase, xylosidase, glucoamylase (AE9),alpha-amylase (AE7), isopullulanase (SE35), alpha-glucosidase (AE10),acetylxylan esterase (T. reesei AXE), and pectinase.

The enzymes that had the best secreted activity in yeast were combinedand screened for the best synergy with each other. FIGS. 43-45demonstrate examples of screening enzymes in combination. Severalamylolytic enzymes were screened for synergy with AE8 by Starch-DNS,Corn Mash and Fiber assays. Supernatants of strains grown for 3 days inYPD were mixed with supernatant with AE8 at 50:50 ratio. In the firstsample of FIG. 43, AE8 supernatant was 100%. Supernatant of M0509 hoststrain was used as negative control. FIG. 43 shows that several AAs andSE11 glucoamylases had positive effect on glucose release when added toAE8 compared to when additional AE8 added. AE7 alpha-amylase hadparticularly strong effect. FIG. 44 shows that on corn mash SE14alpha-glucosidase had positive effect on glucose release when combinedwith AE8.

The effect of arabinases and xylanases on glucose release from nonpretreated corn fiber in the presence of AE8 was also analyzed (FIG.44). FIG. 44 shows that Arab had positive effect on glucose release fromfiber. Several xylanases also had some effect on glucose release fromfiber when added to AE8 (FIG. 45). The information obtained from thescreening of enzyme combinations was used to select the optimal set ofenzymes for a particular substrates such as corn mash, pretreated cornfiber and corn syrup.

Example 21: Screening Industrial Strains for High Ethanol Yield andHeterologous Protein Production

In order to choose the industrial host strain for engineering amylasesseveral industrial and Mascoma developed strains were screened forproduction of ethanol from liquefied corn mash in the presence ofstandard dose of commercial glucoamylases (data not shown). Two of thebest performing strains, M0212 which is a well established highperformance ethanologen, and M0139 which is a high performanceethanologen from the distillery industry, were chosen for furtherevaluation. Since success of the CBP process is dependent on sufficientexpression of heterologous genes in an industrial yeast strain, thestrains were compared for their ability to express amylases. Threestrains were evaluated: two strains selected for high ethanol yield,M0212 and M0139, and M0749—a Mascoma robust strain that does not achievethe ethanol titers of M0212 and M0139 but is known to produce highlevels of heterologous proteins (McBride et al., WO 2010/06000056,2010). The activity levels of three different glucoamylases (AE3, AE8,and AE49) were measured in culture supernatants of the above strainswhen expressed from a multicopy 2μ pMU1575 plasmid. The results areshown in FIG. 46 using maltose as the substrate. Similar results wereobtained using starch (data not shown). The results clearly show thatexpression is lowest when M0212 is the production platform for allenzymes tested. However, strain M0139 served as the best secretionplatform and is also a comparable ethanologen to M0212. A similar trendwas also observed when an alpha amylase (SE15) was expressed in allthree strain backgrounds and activity was measured on starch. Based onthese results M0139 strain was selected as host background strain forengineering CBP strains.

Example 22: Engineering of Marker Free Stable Amylolytic Strains inIndustrial Background

Two approaches were utilized to engineer strains expressing amylolyticenzymes: random integration and directed integration. In both cases thegenes were stably integrated into the genome. When using a radonintegration approach, amylolytic genes were integrated into delta sitesby selection of a linked auxotrophic marker. Several genes wereintegrated at the same time in different combinations and transformantswere screened on starch containing URA—plates. When the directedintegration approach was used, the genes were integrated into designatedloci. Both approaches are described in more details below.

Construction of Strains by Random Integration

In order to study the potential of random integration and the starchplate selection approach for strain construction, four integrativeconstructs with the most active amylolytic enzymes were built (FIG. 47,top). The constructs contain alpha-amylase, 2 glucoamylases, andalpha-glucosidase under different promoters and terminators attached toURA3 marker and flanked by delta integration sites. The constructs weremixed at equal amounts in 7 different combinations (FIG. 47, bottom) and3 μg of total DNA was transformed into industrial strains M1744 (M0139background) and M0749 (M0509 background). Transformants were plated onSD-URA plates and on YM-Starch plates (1×YNB plus 0.5% starch). It wasfound that starch selection without additional marker works for strainswith M0509 strain background but does not in M0139 background strains.Nevertheless the combination of starch and URA selection worked forM0139 strains (a large number of background colonies are obtained ifonly starch used as marker for M0139 strains). The transformantsselected from both kinds of plates and in both host backgrounds werescreened by Starch-DNS assay. The top hits were tested again induplicates twice (FIG. 48). As a result several strains were made withhigh secreted activity on starch. The combinations that made the strainswith the highest activity included: AE9 alone, AE8+AE7, AE9+AE10, andAE9+AE7.

Construction of Strains by Directed Integration

The directed integration approach creates transgenic strains withintegration events that are easier to characterize. Any mistargetingevents can be easily identified with a Southern blot. Additionally,strains engineered by directed approach are potentially more stablesince each expression cassette at the chromosome is integrated into aunique site (not tested). URA3 and FCY1 negative selection approacheswere both developed. FCY1 was eventually chosen as the marker of choicesince fcy mutation did not effect robustness of the strains. Using thistechnology, many clean strains were built in the industrial strainbackground. FIG. 49 demonstrates how glucoamylase expression cassetteswere integrated into FCY1 locus. In this case, counter selection for theFCY1 knock out also selects for integration of the glucoamylaseexpression cassette. In the expression cassettes, the glucoamylase genesare under control of a strong promoter from various central metabolismgenes. When multiple copies are used, the expression cassettescontaining the same sequences are oriented toward each other to decreasethe chance of spontaneous recombination. The glucoamylase expressioncassettes were transformed into industrial strain M0139 as PCR productswith homologous ends targeting the upstream and downstream regions ofthe FCY locus. Since removal of both copies of FCY is necessary forresistance to 5-fluorocytosine (5-FC), each expression cassette wasfound to be integrated on both chromosomes. A 2-μ plasmid, whichcontains a cassette to expresses the Hygromycin resistance gene marker(Hyg), was co-transformed with the PCR products. The transformants werefirst cultivated in liquid YPD+Hyg (300 ug/ml) media overnight and thenplated on media containing 5-fluorocytosine. Precultivation on mediawith antibiotic increases efficiency of double FCY1 knock-out. Thisapproach was also utilized with other negative selection markers such asURA3. Genetic manipulations at the FCY locus result in strains that aremarker free and can be easily modified by recycling the FCY marker. Forinstance, additional copies of AE8 and AE9 could be placed at otherloci.

FIG. 50 demonstrates how more glucoamylase copies could be integratedinto another site such as an Adenine-phosphoribosyltransferase 2 (APT2)locus. In the first round of transformation four additional GAexpression cassettes are amplified by PCR with homologous tails for eachother and a region upstream and down stream of the APT2 locus. Dominantmarkers (Nat and Kan) and the FCY1 marker were integrated into APT2locus into industrial strain M1973 (already expressing 4 GA copies, FIG.49) as PCR products with overlapping ends together with 4 additionalGAs. The transformants were plated on YPD+Nat+Kan plates that allowgrowth of cells that have both dominant markers integrated on thechromosome. Transformants were screened for the high amylolytic activityby Starch-DNS assay. The strain demonstrating the highest activity waschosen and the Kan and Nat markers were removed by transformation of twoPCR products that have homologous ends for each other, the APT2 upstreamflanking region and the 5′—part of AE9 expression cassette. Thetransformants were plated on 5-fluorocytosine containing media thatselects for strains that have lost FCY1. In this approach, expressioncassettes can be integrated into any yeast site as long is the eventdoes not perturb an essential function. The strains with the highestactivity on starch were evaluated further by corn mash fermentation inbioreactors.

Example 23: Evaluation of Amylolytic Strains by Corn Mash Fermentation

Several amylolytic CBP strains that demonstrated the highest activity inscreening assays were evaluated for their ability to produce ethanolfrom liquefied corn mash. The strains used for this experiment werebuilt by either directed or random integration and express differentcombinations of amylases from Saccharomycopsis fibuligera (Tables 18,19). Background non-amylolytic M0139 strain was used as control.Fermentations were performed in sealed shake flasks on corn mashobtained from Valero bio-refinery at 30% solids (TS) at a fermentationtemperature of 32° C. at a shaking speed of 125 rpm. The fermentationswere performed using 500 ppm urea as the only nutrient source. Standarddose (0.45 AGU/g TS) of commercial glucoamylase glucoamylase (SpirizymeUltra, Novozymes) was added to the control strain M0139. All otherstrains were fermented without any exogenous enzymes added. The ethanolproduced after 60 hours of fermentation shown in FIG. 52. FIG. 52 showsthat all CBP strains produced ethanol in an amount similar to thecontrol strain with full dose of glucoamylase. The T6-2 strain producedthe same amount of ethanol in 60 hrs without any added enzymes ascontrol strain M0139. This is the first demonstration of full CBP effectdemonstrated at commercial ethanol production level, when yeast producedenzymes completely replaced exogenous enzyme added in standardcommercial process.

TABLE 18 Description of strains used for fermentation in FIG. 52. Thegenes AE8, AE9, and AE10 described in Tables 16 and 19. StrainDescription M0139 Non-CBP strain with full commercial dose ofGlucoamylase (GA) M1973 Directed Integration (DI) of 2AE8, 2AE9 at FCYsite M2016 Directed Integration (DI) of 4AE9 at FCY site M2022 DI ofMO1973 with 4 copies AE8 and 4 copies AE9 at APT2 site T6-2 RandomIntegration (RI) of AE9 and AE10 at delta sites

TABLE 19Protein and DNA sequences of amylases used to build CBP strains. SeqSeq# Name Gene Source Protein DNA 1 AE1 Gene was obtained byMQISKAALLASLAALVY atgcaaatttcaaaagctgctttgcttgcctcatt PCR withAQPVTLFKRETNADKW ggctgcccttgtttatgctcaaccagtgactctat SaccharomycopsisRSQSIYQIVTDRFARTD tcaaaagagaaactaatgctgataaatggag fibuligeraGDTSASCNTEDRLYCG atcacagtctatttatcaaattgtcactgacaga genomic DNA asGSFQGIIKKLDYIKDMG tttgctagaaccgatggtgatacaagtgcttcct templateFTAIWISPVVENIPDNTA gtaacacagaagatagactttactgtggtggtt (ATCC#9947)YGYAYHGYWMKNIYKI ctttccaaggcatcataaagaagttggattaca NENFGTADDLKSLAQEtcaaagatatgggctttactgctatttggatttctc LHDRDMLLMVDIVTNHcagttgttgaaaacattcccgataacacagca YGSDGSGDSIDYSEYTtatggttatgcttatcatggttactggatgaaga PFNDQKYFHNYCLISNYacatatacaaaattaatgaaaactttggtactg DDQAQVQSCWEGDSSctgatgatttgaagtctttggcacaagaattgca VALPDLRTEDSDVASVFcgatcgtgatatgttgttaatggtcgatatcgtta NSWVKDFVGNYSIDGLccaaccattacggcagtgatggcagtggaga RIDSAKHVDQGFFPDFtagtatcgattactcagagtacaccccgttcaa VSASGVYSVGEVFQGDcgaccaaaagtacttccataactactgtcttatt PAYTCPYQNYIPGVSNtcaaactatgatgaccaagctcaggttcaaag YPLYYPTTRFFKTTDSSttgctgggaaggtgactcttcagttgcattacca SSELTQMISSVASSCSDgatttgagaacggaagatagcgacgtggcct PTLLTNFVENHDNERFAcagttttcaattcttgggttaaagattttgttggca SMTSDQSLISNAIAFVLLattactcaattgatggtttaagaattgatagtgct GDGIPVIYYGQEQGLSaaacatgtggaccaaggctttttcccggattttg GKSDPNNREALWLSGYttagtgcatctggagtttactcagtaggcgaagt NKESDYYKLIAKANAARtttccaaggagacccagcttatacatgcccata NAAVYQDSSYATSQLSccaaaattacattccaggggttagtaattatcc VIFSNDHVIATKRGSVVattgtactacccaaccacgagattttttaaaact SVFNNLGSSGSSDVTISactgattcaagttccagtgagttgactcaaatg NTGYSSGEDLVEVLTCatttcaagcgttgcttccagttgttcggatccaa STVSGSSDLQVSIQGGctttgttgacaaactttgtagaaaatcacgataa QPQIFVPAKYASDICStgaaaggttcgcttcaatgaccagcgaccaa agtttgatttctaatgctattgcatttgtccttttgggtgatggtattcctgtcatttactatggacaagaa caaggcttgagcggaaaaagtgacccaaacaacagagaggccttgtggttatccggctacaa caaagagagtgactattacaagctcattgccaaagctaatgctgccagaaacgccgccgtttat caagactcaagctatgccacctcgcagctttctgtgatcttttcaaatgaccatgttattgcaacaa aaagaggcagcgttgtttctgttttcaacaaccttggttccagcggttcttctgatgtgactatttcca acacaggttacagttccggtgaggatttggtagaagttttgacatgcagtactgttagcggcagct ctgacttacaagtttctatccaaggtggtcaaccacaaatctttgttcctgctaaatatgcttctgac atttgttca 2 AE7 Gene was obtained byMKFATILSTTALALSSLV atgaaatttgcaactatcttaagtacaactgctc PGR withASKPIFLSKRDAGSSAA ttgcgctatcaagtttggttgcatccaagccaat DebaryomycesAAWRSESIYQLVTDRF tttcttaagcaaaagggatgctggcagctctgc occidentalis genomicARTDGSTSATCNTGDR tgctgcagcttggcgttctgaatctatctatcaa DNA as templateVYCGGTFQGIIDKLDYI cttgttaccgatagatttgccagaactgacgga (ATCC#26077)QGMGFTAIWISPWEQI tcgacttcagctacttgtaatactggagataga PDDTGYGYAYHGYWMgtatactgtgggggtactttccaaggtattattg KDIYAINSNFGTADDLKacaaattggattacatccaaggtatgggtttca NLSNELHKRNMKLMVDctgctatttggatttctccagttgttgaacaaattc IVTNHYAWNGAGSSVActgatgatactggttatggttatgcttaccacgg YSNYNPFNQOSYFHDYctattggatgaaagatatttacgctataaattca CLITNYDDQTNVEDCWaattttggtactgccgatgacttgaagaatctttc EGDNTVSLPDLRTEDSaaatgaattgcataagagaaatatgaagctta DVSSIFNLWVAELVSNYtggttgatattgttactaaccattatgcttggaat SIDGLRIDSAKHVDESFggtgccggtagcagtgttgcttactccaactac YPSFOSAAGVYLLGEVaatccattcaaccaacaatcctacttccacgat YDGDPAYTCPYQNYMStattgtttaattacaaattacgatgatcaaacca GVTNYPLYYPMLRFFQatgttgaagattgctgggaaggcgataatact GTSNSVDELNAMISSLEgttagtttaccagatcttcgtactgaggattcag SDCKDITLLGNFIENHDatgttagctctattttcaatctgtgggttgctgagtt QPRLPSYTSDSALIKNAIagtttdaattactcaattgatggtttaaggattg AFNLMSDGIPIIVYGQEacagtgctaagcatgttgatgaatcattctacc QGYSGSSDPNNREALcatcattccaaagtgctgcaggtgtctatcttctt WLSGYSTSNGYYKLISSggagaagtttatgacggtgatccagcttacact VNQIRNQAIYKDSKYTTtgcccataccaaaactatatgtcaggggttact YWSDVLYASGHVIALQaactatcctttgtactatccaatgttaagattcttt RGADDQRIVSVFNNLGcaaggtacttctaactctgtcgatgaattaaatg SSGSQTVTFSTKYSGGctatgatttcaagtttagaaagtgattgtaagga EKVVDVLTCQTSYANStattactttattgggtaatttcattgaaaaccatg DSTLTVSISGGAPRIYAatcaaccaagattaccatcttatacttctgatag PASLIANSGICNFtgccttaatcaaaaatgcaattgcgtttaatttaa tgtcagatggtattccaattatttactacggtcaagaacaaggttacagtggtagctccgatccaa acaacagagaagcattatggttatctggttacagcactagtaatggttactacaaacttatctcttc agttaatcaaattagaaaccaagccatttataaggatagcaaatacactacttattggagtgatgt gttatacgcttcaggtcatgttattgctcttcaaagaggtgcagacgaccaaagaattgtttctgtct ttaacaatttaggctcaagcggatctcaaactgtaacattcagtactaaatacagcggtggagaa aaagtcgttgacgttttaacttgtcaaacttcatacgccaactcggatagtactttaactgtctctatt agtggtggcgctccaagaatttatgctcctgcttctcttattgcaaattctggaatttgcaacttc 3 AE8 Gene was obtained byMRFGVLISVFAAIVSALP atgagattcggtgttttaatctccgtctttgctgct PCR withLQEGPLNKRAYPSFEA attgttagtgctttacctttgcaagaaggtcctttg SaccharomycopsisYSNYKVDRTDLETFLDK aacaaaagagcctatccttcttttgaagcttatt fibuligeraGKEVSLYYLLQNIAYPE caaactataaagttgacagaactgacttggaa genomic DNA asGQFNNGVPGTVIASPS accttcttggacaaacaaaaagaagtatcttta templateTSNPDYYYQWTRDSAI tactatcttttacaaaacattgcttatcctgaagg (ATCC#9947)TFLTVLSELEDNNFNTT ccaatttaataatggtgttcctggtactgttattgc LAKAVEYYINTSYNLGRttctccatcaacctctaatccggactactattac TSNPSGSFDDENHKGLcaatggaccagagattccgcaattacatttttg GEPKFNTDGSAYTGAWacagttctttctgaactagaagataataacttca GRPQNDGPALRAYAISataccactttggccaaggcagttgagtactac RYLNDVNSLNEGKLVLTattaacaccagttacaaccttcaaagaacca DSGGINFSSTEDIYKNIIgtaacccaagtggcagctttgatgatgaaaat KPDLEYVIGYWDSTGFcataaaggcttgggagaaccaaaatttaaca DLWEENQGRHFFTSLVcagatggttctgcatacaccggagcttggggg QQKALAYAVDIAKSFDDagaccgcaaaatgatggtcctgctttgagagc GDFANTLSSTASTLESYttatgctatcagtagatacttgaatgatgtcaatt LSGSDGGFVNTDVNHIctttaaatgaaggtaaattagtattgactgattc VENPDLLQQNSRQGLDaggtggtatcaacttttcttcaactgaagatattt SATYIGPLLTHDIGESSSacaaaaatatcatcaaaccagacttggaatat TPFDVDNEYVLQSYYLLgttatagggtactgggattctactgggtttgatct LEDNKDRYFVNSAYSAttgggaggaaaaccaaggcagacactttttta GAAIGRYPEDVYNGDGcaagcttggttcaacagaaagcccttgcttatg SSEGNPWFLATAYAAQctgtcgatattgccaaaagttttgacgacggcg VPYKLAYDAKSASNDITIactttgcgaacacactttcttcgactgcttctacc NKINYDFFNKYIVDLSTIctcgaaagttatttgagtggcagtgatggtgga NSAYQSSDSVTIKSGStttgttaatactgatgttaaccacattgttgaaaa DEFNTVADNLVTFGDScccagatttgcttcaacaaaactctagacaag FLQVILDHINDDGSLNEgtctagattcagccacatatattggcccactttt QLNRYTGYSTGAYSLTgactcatgatattggtgaaagcagctcaactc WSSGALLEAIRLRNKVKcatttgatgttgacaatgagtatgttttgcaatca ALAtattacttgttattggaggataacaaagacaga tactttgttaacagtgcttattctgctggtgcagctattggcagatacccagaagatgtttacaatggt gatggttcatctgaaggcaatccatggttcttagctactgcctatgctgcccaagttccatacaaac ttgcttatgatgcaaagtcggcctcaaatgacattaccattaacaagattaactacgatttttttaacaagtatattgttgatttatctaccatcaattctgctt accagtcttctgatagtgtcaccattaaaagtggctctgatgaatttaacacggttgctgataatttg gtcacattcggtgattcctttttgcaagtcattttggatcatattaatgatgatggctccttgaatgaac aacttaacagatataccggttattccaccggtgcctactctttgacatggagcagtggtgctcttctt gaagctattagacttagaaataaggtcaaggctttggcttaa 4 AE9 Gene was codon MIRLTVFLTAVFAAVASatgatcagattgaccgttttcttgaccgctgttttt optimized for CVPVELDKRNTGHFQAgctgctgttgcttcttgtgttccagttgaattggat S. cerevisiae and YSGYTVARSNFTQWIHaagagaaacaccggtcatttccaagcttattct synthetized by EQPAVSWYYLLQNIDYggttataccgttgctagatctaacttcacccaat GeneArt PEGQFKSAKPGVVVASggattcatgaacaaccagctgtttcttggtacta (PubMed#CAC83969.1) PSTSEPDYFYQWTRDTcttgttgcaaaacatcgattacccagaaggtc AITFLSLIAEVEDHSFSNaattcaaatctgctaaaccaggtgttgttgttgct TTLAKVVEYYISNTYTLtctccatctacatctgaaccagattacttctacc QRVSNPSGNFDSPNHDaatggactagagataccgctattaccttcttgtc GLGEPKFNVDDTAYTActtgattgctgaagttgaagatcattctttctcca SWGRPQNDGPALRAYacactaccttggctaaggttgtcgaatattacat AISRYLNAVAKHNNGKLttccaacacctacaccttgcaaagagtttctaat LLAGQNGIPYSSASDIYccatccggtaacttcgattctccaaatcatgatg WKIIKPDLQHVSTHWSTgtttgggtgaacctaagttcaacgttgatgatac SGFDLWEENQGTHFFTtgcttatacagcttcttggggtagaccacaaaa ALVQLKALSYGIPLSKTtgatggtccagctttgagagcttacgctatttcta YNDPGFTSWLEKQKDAgatacttgaacgctgttgctaagcacaacaac LNSYINSSGFVNSGKKHggtaaattattattggccggtcaaaacggtattc IVESPQLSSRGGLDSATcttattcttctgcttccgatatctactggaagatta YIAALITHDIGDDDTYTPttaagccagacttgcaacatgtttctactcattg FNVDNSYVLNSLYYLLVgtctacctctggttttgatttgtgggaagaaaatc DNKNRYKINGNYKAGAaaggtactcatttcttcaccgctttggttcaattg AVGRYPEDVYNGVGTSaaggctttgtcttacggtattccattgtctaagac EGNPWQLATAYAGQTFctacaatgatccaggtttcacttcttggttggaa YTLAYNSLKNKKNLVIEaaacaaaaggatgccttgaactcctacattaa KLNYDLYNSFIADLSKIDctcttccggtttcgttaactctggtaaaaagcac SSYASKDSLTLTYGSDatcgttgaatctccacaattgtcatctagaggtg NYKNVIKSLLQFGDSFLgtttggattctgctacttatattgctgccttgatca KVLLDHIDDNGQLTEEIcccatgatatcggtgatgatgatacttacaccc NRYTGFQAGAVSLTWScattcaatgttgataactcctacgttttgaactcc SGSLLSANRARNKLIELttgtattacctattggtcgacaacaagaacaga L tacaagatcaacggtaactacaaagctggtgctgctgttggtagatatcctgaagatgtttacaa cggtgttggtacttctgaaggtaatccatggcaattggctactgcttatgctggtcaaactttttaca ccttggcctacaattccttgaagaacaagaagaacttggtcatcgaaaagttgaactacgacttg tacaactccttcattgctgatttgtccaagattgattcttcctacgcttctaaggattctttgactttgacc tacggttccgataactacaagaacgttatcaagtccttgttgcaattcggtgactcattcttgaagg ttttgttggatcacatcgatgacaacggtcaattgactgaagaaatcaacagatacaccggttttc aagctggtgcagtttctttgacttggtcatctggttctttgttgtctgctaatagagccagaaacaagtt gatcgaattattg 5 AE10 Gene was codonMIWLKLSLYSLAFALFA atgatctggttgaagttgtccttgtactctttggctt optimized forDAAPVSSGEEAETSSS ttgctttgtttgctgatgctgctccagtttcttctggtS. cerevisiae and TSSSAPAQITVDNELTL gaagaagctgaaacttctagctctacttcttcatsynthetized by GVSQVPNIVNKTAIDAN ctgctccagctcaaattaccgttgataacgaatGeneArt EAAKGVDLVNVTTTAK tgaccttgggtgtttctcaagttccaaacatcgtt(Pub Med#CAF31354.1) GLTGILKLNEATNIYGY aacaagaccgctattgatgctaatgaagctgcDFDYLNLSVEYQSDDR taaaggttacgatttggttaacgttactactactg LNVHIEPVDTDNVFILPEctaagggtttgaccggtattttgaagttgaatga SLVAKPSADDGDKIESFagccactaacatctacggttacgatttcgatta HFGGSSDLVFEYSSKNcttgaacttgtccgtcgaataccaatccgatga FGFEILRKSTGKSIFSTItagattgaacgttcacatcgaaccagttgatac GNPLVFSNQFIQFNTSLcgataacgttttcattttgccagaatccttggttg PKDHFITGLGESIHGFRctaaaccatctgctgatgatggtgataagatcg NEPGIVKTLYANDIANPIaatcttttcatttcggtggttcctccgatttggttttt DGNIYGVHPFYIDQRFDgaatactcttccaagaacttcggtttcgaaatct TNATHGVYWRTSAIQEtgagaaagtctaccggtaagtctattttctccac VAVGNESLTWRALSGItattggtaacccattggttttctccaatcaattcat VDLYFFSGPKPKDVIQQccaattcaacacatccttgccaaaggatcattt YVKEVGLPTFQPYWALcattactggtttgggtgaatccatccatggtttta GYHQCRWGYDTIEELDgaaatgaaccaggtatcgtcaaaaccttgtac EVVENFKNFDIPLETIWgctaatgatattgccaacccaatcgatggtaat SDIDYMDSYKDFTNDPatctatggtgttcacccattctacatcgatcaaa HRYPLEKYQQFLDKLHgatttgataccaacgctacccatggtgtttattg ENNQHYVPIIDAAIYVPNgagaacttctgccattcaagaagttgctgttggt PENATDNDYDVFHYGNaacgaatccttgacttggagagctttgtctggta ETDVFLKNPDGSLYIGAtagttgacttgtactttttctccggtccaaaacct VWPGYTVFPDFLSENIaaggatgtcattcaacaatacgtcaaagaagt QKYWTKVFKDWYQQIKtggtttgccaacttttcaaccatattgggctttgg FDGIWLDMNEVSSFCVgttaccatcaatgtagatggggttacgatacca GSCGSGKITDNPVHPPtcgaagaattggatgaagtcgtcgaaaacttc FAVGGEATEFPEGFNKaagaacttcgatattccattggaaaccatctgg TNGTEYASFTSSLAAAStccgatatcgattacatggattcaacaaggatt PTSDEDSSASSTSASIDtcaccaacgatccacatagatacccattggaa SLNTLAPGKGNINYPPYaagtaccaacaattcttggacaagttgcacga AINNDQGDHDLATHAVaaacaatcaacactacgttccaattattgatgc SPNATHQDGTLEYDVHcgctatctacgttccaaatccagaaaatgctac NLYGYLETNATFEALLEIcgataacgattacgatgttttccattacggtaac QPNKRPFIISRSSFAGSgaaaccgacgtttttttgaagaatccagatggtt GRQTGHWGGDNYSQFccttgtacattggtgctgtttggccaggttatact RSAYFSIAQAFSFGLSGgtttttccagatttcttgtccgaaaacatccaaa IPFFGADVCGFNGNSDagtactggaccaaggttttcaaggactggtatc YELCSRWMQLGSFFPFaacaaatcaagttcgatggtatctggttggata YRNHNILGAISQEPYVWtgaacgaagtttcttctttctgtgttggttcttgtggt ESVTEATKTSMQIRYLLtctggtaagattactgataacccagttcatcca LPYYYTLLHEAHITGIPILccatttgctgttggtggtgaagctactgaatttcc RAFAWQFPENKNVSTVagaaggtttcaacaagaccaacggtactgaa DTQFFVGDALVVTPALEtacgcttctttcacttcttctttggctgctgcttctcc QGVDTVKGTFPGSGNEaacttctgatgaagattcttctgcttcttctacctct EVYYDWYTHEKQNFTDgcttctattgattctttgaacactttggctccaggt GKNETLQAPLGHIPLHIaagggtaatattaactatccaccatacgccat RGGHILPTQEPAYTTTEcaacaacgatcaaggtgatcatgatttggcta SRQNPWGLIVALDKDGctcatgctgtttctccaaatgctactcatcaagat KAEGKLYSDDGESYEVggtactttggaatacgatgtccataacttgtacg EESLFVNFIASDNTLLSTgttactttggaaactaacgctactttcgaagcctt SYGEYEVEQPLANITILgttggaaatccaacctaacaaaagaccattc GVENKPKEVKFDDSKVatcatctccagatcttcatttgctggttctggtag DFTFENNTIFVTGLDDQacaaactggtcattggggtggtgataattactct TEDGAFAKHFKLSWcaattcagatctgcctacttctctattgctcaagctttttctttcggtttgtccggtattccattttttggtgctgatgtttgtggtttcaacggtaattccgattacga attgtgttccagatggatgcaattgggttcatttttcccattctacagaaaccacaacattttgggtgc catttctcaagaaccatacgtttgggaatctgttactgaagctactaagacctccatgcaaatca gatatttgttgttgccttactactacaccttgttgcatgaagctcatattaccggtatcccaattttgag agcttttgcttggcaattcccagaaaacaagaacgtttctaccgttgatacccaattctttgttggtg atgctttggttgttactccagctttggaacaaggtgttgatactgttaagggtacttttccaggttctggt aacgaagaagtttactacgattggtacacccacgaaaagcaaaatttcactgacggtaagaac gaaacattgcaagctccattgggtcatattccattgcatattagaggtggtcatatcttgccaactc aagaaccagcttacactactactgaatctagacaaaatccatggggtttgatagttgccttggata aggatggtaaagccgaaggtaaattatactccgatgatggtgaatcctacgaagttgaagaatc cttgttcgttaacttcattgcttccgataataccttgttgtctacctcttacggtgaatatgaagtcgaa caaccattggccaacattactattttgggtgttgaaaacaagccaaaagaagttaagttcgacg attccaaggttgatttcaccttcgaaaacaacaccattttcgttaccggtttggatgatcaaactga agatggtgcttttgctaagcactttaagttgtcttgg

Example 24: Evaluation of CBP Strains Performance on Raw Corn Mash

The performance of selected CBP strains was also evaluated byfermentation of non-liquefied corn starch (FIG. 53). FIG. 53demonstrates that even though the sets of enzymes expressed in those CBPstrains were not optimized for this substrate, over 80 g/l ethanol wasproduced by CBP strains from raw mash in 72 h without any exogenousenzymes.

Example 25: Improving Strain Performance by Evolution

Yeast is known for its ability of adjustment to very broad range ofconditions. This property could be used to increase yeast ethanol andhigh temperature resistance and improve performance (ethanol yield) atcertain relevant conditions such as during fermentation of corn mash. Toexplore this possibility as a tool to develop better CBP yeast strainsthat are able to reach higher ethanol yield, one of the best CBP strainsM1973 was evolved by using serial transfer in corn mash. Serial transferfermentations were carried out using shake flasks containing 35% TSliquefied corn mash with industrial medium grown at 35° C. and 150 rpm.At 3 days intervals, 10 ml were transferred to fresh medium of the samecomposition (5 transfers). At each transfer starting with the second thetemperature was raised 1 degree. At the last transfer it was 38° C.After 5 transfers (˜500 hours), the cell were plated on YPD plates forevaluation. The evolved strain was evaluated by fermentation onliquefied corn mash at two different temperatures (32° C. and 35° C.)and two different concentrations of solids (30% and 35%). Original M1973strain from the freezer stock was used as control (FIG. 54). FIG. 53demonstrates that at all conditions tested adapted M1973 strain was ableto produce more ethanol than parental M1973 at 48 hrs. Thereforeevolution of yeast strains was proven to be a powerful tool fordeveloping better strains.

Example 26: Process Flow Sheet with CBP Strains

The example of CBP process in presented on FIG. 55. In this example twoyeast CBP strains are used in the process and cultured separately, S1and S2. Liquefied corn pre-treated with alpha-amylases is fermented byyeast strain S1. S1 has optimal set of amylases and accessory enzymesengineered to efficiently convert corn starch into glucose without anyexogenous enzymes added. After ethanol distillation the stillage isbeing pre-treated and fermented by strain S2. S2 has cellulolytic set ofenzymes engineered and optimized for corn fiber conversion as well asxylose and arabinose pathways. S2 also has amylolytic enzymes engineeredbecause more starch is being released while corn fiber pretreatment.Ground raw corn mash could also be utilized. In this case noalpha-amylase pre-treatment is necessary and alpha-amylase could beexpressed by strain S1.

Example 27: Screening and Characterization of Industrial Yeast Strains

The objective of this study was finding an industrial host that willcombine high temperature/ethanol tolerance and high heterologous proteinsecretion. Several industrial yeast strains were obtained from variouscommercial sources (Table 20). In order to better understand thestrains' relations with each other, all strains were genotyped asdescribed by Ness et al., 1993 (FIG. 56). The similarity between bandpatterns or genotyping patterns reflects strain's genetic similarity.Most of strains demonstrated one of 2 genotyping patterns. One patternwas similar to M0139 and other was similar to M2390. The pattern ofM2392 was different from others.

The industrial strains were compared for their ability to grow at hightemperature (FIG. 57). FIG. 57 shows that the strains demonstratedsignificantly different growth at 41° C. The same pattern was confirmedwhen 41° C. maximum growth rate in YPD was measured quantitatively byplate reader (FIG. 58, top). The strains were also tested forrobustness—maximum ethanol titer reached on high solids with full enzymedose (FIG. 58, bottom). A comparison of the maximum growth data 41° C.with robustness data reveals that there is a positive correlationbetween high temperature tolerance and high ethanol tolerance.Therefore, the ability of strains to reach high ethanol titers could beestimated by their 41° C. maximum growth rate in high throughput format.The data shown in FIGS. 56 and 58 are summarized in Table 21. The datain Table 21 demonstrate that strains from ethanol industry (genotypingpattern B) tend to have higher ethanol and high temperature tolerancecompared to wine strains (genotyping pattern A).

In order to compare ability of industrial strains to expressheterologous proteins, the host strains from Table 20 were transformedwith the same expression construct of AE9—Saccharomycopsis fibuligeraglucoamylase gene (Accession No. CAC83969.1). Four copies of AE9 weredirectly integrated into FCY locus. FCY was used as negative marker. Theconstruct used was similar to the one used for M2016 construction(Example 23). The map of the expression construct used in thisexperiment shown on FIG. 60. Several transformants for each host werepicked and screened for starch activity (FIG. 59). Different hoststrains demonstrated different ability to secrete GA. Interestingly, twobatches of the same strain, M0212 and M2390, had different averageexpression level of the same AE9 expression construct. Thus, it wasdemonstrated that robust ethanol tolerant hosts from ethanol industrylike M2390 can be suitable host for engineering CBP strains.

Transformants for each host that were the most active on starch (Table22) were tested in shake flask fermentation on raw corn flour andconventional corn mash together with non-transformed hosts (FIG. 61-62).FIGS. 61 and 62 demonstrate that both host strains M0212 and M2390 andtheir GA transformants, M2395 and M2399, have superior performance onboth tested substrates compared to other tested industrial strains.M2390 had higher average GA expression/secretion level than M0212 andtherefore was chosen as the host to engineer CBP strains.

TABLE 20 Industrial ethanologen strains used in the study. StrainMascoma# name Producer Reference M139 N96 Anchor winewww.anchorwineyeast.com/pdf/ yeast N 96.pdf M212 Ethanol LaSaffrewww.lesaffreyeastcorp.com/ Red (old) home/ M2390 Ethanol LaSaffrewww.pahc.com/Phibro/ Performance-Products/Catalog/ Red (new)23/Ethanol-Red.html M2394 FALI ABMauri www.alcoholyeast.com/downloads/doc1.pdf M2393 Premier LaSaffre mountainhomebrew.com/premiercuvee- Cuvee 5grampackage.aspx M2392 Lalvin ICV- Lallemandwww.lalvinyeast.com/images/ K1 library/ICV-K1_Yeast.pdf M2391 Lalvin EC-Lallemand store.homebrewheaven.com/ 1118 lalvin-ec-1118-champagne-wine-yeast-p1076.aspx M2507 NABC Bio- North www.na-bio.com/ Ferm XR Americaindex.php?option= com_content&view= Bioproducts article&id=74&Itemid=263

TABLE 21 Summary of industrial strains screening. The summary is basedon the data shown on FIGS. 56 and 58. EtOH 41C on Main Genotyping growthflour Mascoma# application pattern rate g/l M0139 Wine M139 like 0.03132 M0212 Ethanol M212 like 0.21 141 M2390 Ethanol M212 like 0.16 143M2394 Ethanol M212 like 0.17 143 M2393 Wine M139 like 0.03 132 M2392Wine New 0.08 124 M2391 Wine M139 like 0.18 146 M2507 Ethanol M212 like0.35 147

TABLE 22 Industrial strains transformed with 4 copies ofSaccharomycopsis fibuligera glucoamylase gene (NCBI#CAC83969.1) andtheir most active on starch transformants selected by starch assay (FIG.59). Strain M2111 was made the same way as M2016, only more colonies(84) were screened by starch assay. Several the most active colonieswere screened by industrial corn mash fermentation and the bestperforming strain was named M2111. Host strain Transformant M139 M2400,M2111 M212 M2399 M2390 M2395 M2394 M2398 M2393 M2397 M2391 M2396

Example 28: Increasing Heterologous GA Production by HighEthanol/Temperature Tolerant Yeast Strain

The objective of this study was engineering ethanol/temperature tolerantindustrial yeast strain expressing high level of heterologousglucoamylase. The strain M2111 was made the same way as M2016 (Example23), only more colonies (84) were screened by starch assay. Even thoughit was demonstrated that ethanologen M2390 host has much higherethanol/temperature tolerance compared to wine strain M0139 and performssignificantly better at high solids or high temp conditions whensupplemented with high dose of exogenous enzyme (Example 28), M2111transformant derived from M0139 (Table 22) has much higher AE9 secretionlevel compared to M2395 derived from robust M2390 (FIG. 63). Due to highlevel GA production, M2111 was reaching higher ethanol titer at lowersolids and lower temperature fermentations without exogenous enzymeadded compared to M2395. Therefore it was necessary to increase GAproduction by M2390 host in order to improve CBP performance—maximumethanol reached at low or no exogenous enzyme added. There is asignificant activity variation between transformants even when obtainedwith directed integration. Therefore screening more transformantsusually yields strains with higher expression level. Only severaltransformants were screened when M2395 was selected. In order toincrease AE9 expression level in M2390 host, M2390 was transformed withthe same AE9 expression construct as was used to obtain strain M2016.The expression construct was integrated into FCY locus and FCY was usedas negative selection. About 1000 transformants were screened for starchactivity. Starch assay for the best 30 transformants was repeated intriplicates (FIG. 63). Several transformants demonstrated activitysimilar to M2111 and much higher than M2395.

Seventeen of the most active transformants were screened for CBPperformance by minivial fermentation assay with corn flour and homemademash (FIGS. 64-65). The advantage of new robust background wasespecially noticeable in corn flour fermentation experiment. The newstrains demonstrated significantly better performance compared to lessrobust M2111 strain and reached higher ethanol titers. Several beststrains were further analyzed by shake flask corn flour fermentation(FIG. 66). Results of shake flask fermentation confirmed ability of newrobust CBP strains to reach above 140 g/l ethanol on 33% corn flour with6 times less exogenous enzyme added compared to standard raw corn flourprocess.

Time course fermentation of conventional mash (FIG. 67) and raw cornflour (FIG. 68) was performed for one of the best M2390+AE9transformant—M2691 strain (P10-19). Untransformed host M2390 was used asa control in both experiments. On corn mash, M2691 was fermented withoutany exogenous enzymes added, while standard (for corn mash process) doseof commercial glucoamylase (0.3 AGU/g solids) was added to the controlM2390. On corn flour, standard for raw substrate GA dose (0.6 AGU/g) wasadded to the M2390 and 6 times less enzyme was added to GA expressingM2691 strain. FIG. 67 demonstrates that in conventional liquefied cornmash fermentation process genetically engineered GA producing strain isable to provide complete CBP and reach above 125 g/l ethanol at 72hours. To our knowledge, this is the first time demonstration of highindustrially relevant ethanol titers reached by genetically engineeredstrain without any exogenous enzymes added. FIG. 68 demonstrates that onraw corn substrate GA producing strains can reach even higher ethanoltiter (above 140 g/L at 72 h) which is a standard for raw corn flourfermentation industry. Small dose of exogenous enzyme still needs to beadded to the engineered strain to provide optimal fermentation, butamount of exogenous enzyme added can be decreased several fold.

Example 28: Increasing Heterologous GA Production Effects ExogenousEnzyme Dose Reduction

FIG. 69 demonstrates how amount of GA heterologously produced by yeaststrains effects exogenous enzyme dose reduction. Three strains were usedfor this experiment: untransformed M2390, low GA producer M2395, andhigh GA producer M2519 (P6-65). The strains were fermented on corn flourin shake flasks with different dose of GA added. Standard corn flourindustrial GA dose of 0.6 AGU/g solids was counted as 100%. This dataclearly demonstrate that amount of GA produced by yeast strain hassignificant effect on exogenous GA dose reduction. For the specificexogenous GA used (Spirizyme Ultra) there was at least 75% dosereduction due to heterologously expressed GA by M2519 strain.Furthermore, at the end of fermentation there was extra glucose presentwith GA producing strains. It was shown in other experiments that thisglucose can be transformed into additional ethanol yield at 100%exogenous enzyme dose if fermentation of corn flour performed at lower33% solids.

Example 29: Stability of Glucoamylase Expression

Stability of GA expression was tested for several M2390+AE9 strains.Data for strains M2519 and M2691 are shown in FIG. 70. Strains werepropagated in YPD, grown to stationary phase and passaged with 100×dilution 11 times (1 passage equals about 9 generations). Severalsamples between passages were stocked. All samples and original strainwere plated and inoculated in YPD together. Then activity on starch forall samples was measured in the same starch assay. Out of nine strainstested only three lost some activity (10-50%). Majority of the strainsretained 100% of their amylolytic activity for up to 99 generations.This data indicated that most of strains built by directed integrationare genetically stable.

Example 30: Screening Saccharolytic Genes for Functional Expression inYeast

Multiple genes encoding for saccharolytic enzymes were screened forfunctional expression in yeast (Table 23). The genes were eithersynthesized by GencArt (now Life Technologies) or isolated by PCR fromgenomic DNA. Some genes were expressed with native signal sequences andin others native signal sequence was replaced by S.cerevisiae invertasesignal sequence. Some synthetic genes were codon optimized forexpression in S.cerevisiae (by GeneArt) and others were synthesized withnative DNA sequence. All genes were expressed under ENO1 promoter andterminator from 2-micron plasmid pMU1575. The genes were insertedbetween PacI/AscI sites of pMU1575 either by cloning or yeast mediatedligation. Expression contracts were transformed into an industrialbackground Mascoma strain M1744 and selected on minimal URA deficientmedia. Transformants were grown in YPD for 3 days and supernatants wereanalyzed for activity on starch, pullulan, xylan, pNPX (xylosidaseactivity), maltose and pectin (FIG. 71). The assays for each enzyme werechosen based on predicted activity. The enzymes that demonstratedsecreted activity in one or more assays are in rows bolded in Table 23.FIG. 71 shows results of pullulan, xylan and pectin assays for someenzymes. Isopullulanase SE35 was active on pullulan. Five xylanases wereactive on xylan and three pectin lyases were active on pectin.Pullulanase SE41 had slight secreted activity on pullulan. GlucoamylaseAE82 had some secreted activity on starch and maltose.

TABLE 23 Genes analyzed for functional expression in yeast. For mostgenes protein sequence was obtained from NCBI database. For genes markedwith “* “DNA gene sequence was obtained from MascomaThermoanaerobacterium saccharolyticum genome sequence data. Organ-Signal Codon ID ism Source Enzyme NCBI# Gene sequence optimization SE12Fungi Aspergillus oryzae Glucoamylase BAA01540.1 Synthetic Native NoneSE13 Fungi Rhizopus oryzae Glucoamylase BAA00033.1 Synthetic Native NoneSE28 Fungi Aspergillus niger Xylosidase CAK39870.1 Synthetic Native NoneArabinofuranosidase SE32 Fungi

Xylanase AAS46914.1 Synthetic Native None SE33 Fungi

Xylanase AAS46913.1 Synthetic Native None SE34 Fungi

Xylanase CAA03655.1 Synthetic Native None SE35 Fungi

Isopullulanase BAA19473.1 Synthetic Native None SE37 Fungi Aspergillusniger Endopolygalacturonase XP 001389562.1 Synthetic S.c. InvertaseS.cerevisia SE38 Fungi Aspergillus niger EndopolygalacturonaseCAK42510.1 Synthetic S.c. Invertase S.cerevisia SE41 Plant

Pullulanase NP 001104920.1 Synthetic S.c. Invertase S.cerevisia SE42Plant Olyza sativa Pullulanase ACY56113.1 Synthetic S.c. InvertaseS.cerevisia SE43 Plant Zea mays Isoamylase ACG43008.1 Synthetic S.c.Invertase S.cerevisia SE47 Fungi

Xylanase CAA53632.1 Synthetic Native None SE48 Fungi

Xylanase CAD34597.1 Synthetic Native None

SE49 Fungi Trichoderma viride Xylanase AAQ67413.1 Synthetic Native NoneSE50 Plant Triticum aestivum Pullulanase ABL84490.1 Synthetic S.c.Invertase S.cerevisia SE51 Yeast Saccharomyces Endopolygalacturonase NP012687.1 Native Native cerevisiae SE52 Yeast KluyveromycesEndopolygalacturonase AAR84199.1 Native Native marxianus SE53 BacteriaBacillus subtilis Pectin lyase NP 389746.1 Native S.c. Invertase SE54Bacteria Bacillus licheniformis Polygalacturonase YP 080606.1 NativeS.c. Invertase SE55 Bacteria Bacillus licheniformis Pectin lyase YP079258.1 Native S.c. Invertase SE56 Fungi Aspergillus nigerEndopolygalacturonase CAB72125.1 Synthetic S.c. Invertase S.cerevisiaSE57 Fungi Aspergillus niger Endopolygalacturonase CAB72126.1 SyntheticS.c. Invertase S.cerevisia SE58 Fungi Aspergillus nigerEndopolygalacturonase XP 001390812.1 Synthetic S.c. InvertaseS.cerevisia SE59 Fungi Aspergillus niger EndopolygalacturonaseCAB72931.1 Synthetic S.c. Invertase S.cerevisia SE60 Fungi Aspergillusniger Endopolygalacturonase CAK44164.1 Synthetic S.c. InvertaseS.cerevisia SE61 Fungi

Pectin lyase CAK48529.1 Synthetic S.c. Invertase S.cerevisia SE62 Fungi

Pectin lyase CAK37997.1 Synthetic S.c. Invertase S.cerevisia SE63 FungiAspergillus niger Pectin lyase AAW03313.1 Synthetic S.c. InvertaseS.cerevisia SE64 Fungi

Pectin lyase CAK47350.1 Synthetic S.c. Invertase S.cerevisia SE65 FungiAspergillus niger Pectin lyase ACE00421.1 Synthetic S.c. InvertaseS.cerevisia AE11 Yeast Lipomyces Alpha-amylase AAC49622.1 SyntheticNative None kononenkoae AE79 Yeast Arxula adeninivorans GlucoamylaseCAA86997.1 Synthetic Native None AE81 Fungi Hormoconis resinaeGlucoamylase CAA48243.1 Synthetic Native None AE82 Fungi

Glucoamylase ADN65121.1 Synthetic S.c. Invertase S.cerevisia

AE83 Bacteria Thermoanaerobacterium Polygalacturonase Contig1 GeneNative S.c. Invertase saccharolyticum or0164* AE84 BacteriaThermoanaerobacterium Polygalacturonase Contig1 Gene Native S.c.Invertase saccharolyticum or0344*

Example 31: Identifying Enzymes and their Combinations that IncreaseSugars Release from Industrial Corn Substrates

Distiller corn syrup, which is a soluble fraction left from processingcorn to ethanol, was one of the substrates used to identify enzymes thatwill allow releasing more sugars from corn mash. Corn syrup containssoluble oligosaccharides that are left undigested in corn mashhydrolysis/fermentation process. Several yeast-made enzymes were testedfor conversion of corn syrup. Several enzymes: CBH1, CBH2, EG2, BGL,XYL, and XLD were purified by ion exchange and hydrophobic interactionchromatography on the FPLC from yeast supernatants (Table 24). Forothers yeast strains expressing enzymes were grown for 3 days in YPD andsupernatants were used as enzyme source. Table 24 summarizes theinformation on enzymes used in this experiment. Supernatants of twoenzymes were mixed in equal ratio by volume. Supernatants of singleenzymes were mixed with supernatant of empty strain control M0139. FIG.72 show the results of one of those assays. The experiment was done withand without yeast made glucoamylase (AE9). Table 25 shows how much ofeach purified enzyme was used in this corn syrup assay. Addition of someenzymes increased sugars release from corn syrup. AE9 itself had thebiggest impact indicating that there is a lot of starch left undigestedafter corn mash processing. Other enzymes such as alpha-glucosidase,beta-glucosidase, acetyl xylan esterase (in combination with cellulasesand hemicellulases) also gave essential increase in glucose release fromcorn syrup.

Based on this data, several genes were selected that have a potential toimprove AE9 glucoamylase expressing strain M2111 due to increased sugarrelease from corn mash or corn flour. The selected genes are listed inTable 26. Other candidates in Table 26 were chosen based on a rationalapproach based on which enzymes may have effect on sugar release basedon substrate structure (Saulnier et al., Carbohydrate Polymers, 26:279-287, 1995). All genes selected demonstrated functional expression inyeast.

TABLE 24 Enzymes used in corn syrup assay (FIG. 24). All enzymes exceptAE9 were expressed on 2u plasmid under S. cerevisiae ENO1 promoter andterminator from 2-micron plasmid pMU1575. AE9 in M2111 was expressedfrom 4 gene copies integrated into chromosome (the same as in M2016).The genes were codon optimized for S. cerevisiae and synthesized byGeneArt. Yeast and fungal genes were expressed with native signalsequences. Bacterial gene was attached to S. cerevisiae Invertase signalsequence. ID Strain Source Enzyme Reference Enzyme prep CBH1 Talaromycescellobiohydrolase I WO/2010/060056 HPLC purified emersonii Trichodermareesei CBH2 Chrysosporium cellobiohydrolase II WO/2010/060056 HPLCpurified lucknowense EG2 Trichoderma reesei endoglucanase IIWO/2010/060056 HPLC purified BGL Saccharomycopsis beta-glucosidaseWO/2010/060056 HPLC purified fibuligera XYL Clostridium Xylanase (BC60)NCBI# HPLC purified phytofermentans YP_001558623.1 XLD Pyrenophoratritici- beta-xylosidase NCBI# HPLC purified repentis XM_001940921.1 NCM139 None none none Supernatant AE9 M2111 Saccharomycopsis Glucoamylase(AE9) NCBI# Supernatant fibuligera CAC83969.1 ABF M1511 Aspergillusniger arabinofuranosidase NCBI# AAA93264 Supernatant AXE M1782Trichoderma reesei acetylxylanesterase NCBI# Q99034 Supernatant FAEM1475 Aspergillus niger feruoyl esterase NCBI# Supernatant XP_001393337ARA M2069 Bacillus arabinase NCBI# Supernatant licheniformis AAU41895.1AE10 M1923 Saccharomycopsis alpha-glucosidase NCBI# Supernatantfibuligera CAF31354.1 SE35 M2614 Aspergillus niger isopullulanase NCBI#Supernatant BAA19473.1

TABLE 25 Amounts of purified enzymes used in corn syrup assay experiment(FIG. 72) in mg of enzyme per g of total solids. Load Protein mg/g CBH11.6 CBH2 1.6 EG2 0.6 BGL 0.2 XYL 0.4 XLD 0.2

TABLE 26 Enzymes selected to be expressed in M2111 strain alone or incombinations. Gene ID Source Enzyme AE1 Saccharomycopsis fibuligeraalpha-amylase AE3 Debaryomyces occidentalis alpha-glucosidase AE5Debaryomyces occidentalis alpha-amylase AE7 Debaryomyces occidentalisalpha-amylase AE8 Saccharomycopsis fibuligera glucoamylase AE8 + SBDSaccharomycopsis fibuligera S.f.glucoamylase + SBD of Aspergillus nigerA.n.glucoamylase (SE11) AE9 Saccharomycopsis fibuligera glucoamylaseAE10 Saccharomycopsis fibuligera alpha-glucosidase AE22 Clostridiumphytofermentans pullulanase AE73 (ARA) Bacillus licheniformis arabinaseSE20 Aspergillus niger beta-glucosidase SE32 Aspergillus niger xylanaseSE33 Aspergillus niger xylanase SE34 Aspergillus niger xylanase SE35Aspergillus niger isopullulanase SE39 (ABF) Aspergillus nigerarabinofuranosidase SE47 Humicola insolens xylanase SE48 Talaromycesemersonii xylanase SE66 (AXE) Trichoderma reseei acetyl xylan esteraseSE67 (FAE) Aspergillus niger feruoyl esterase BC60 (XYL) Clostridiumphytofermentans xylanase FAE2 Talaromyces stipitatus feruoyl esteraseSBD—starch binding domain.

Example 32: Construction and Screening of Improved Amylolytic Strains

To make a transformation host for additional AE9 saccharolytic enzymesexpression, URA3 was knocked out of M2111 and the resulting M2125 strainwas used as a host for transformations. For each enzyme from Table 26integrative expression cassette was built targeting delta sites onchromosome. URA3 gene was used as autotrophic selection marker. Eachgene of interest under control of S. cerevisiae strong constitutivepromoter and terminator was inserted between URA3 and Delta2 fragmentsof pMU2382 vector digested with BamHI and EcoRI (FIG. 73). Theexpression cassette was inserted by yeast mediated ligation in the sameorientation as URA3. The fragment that includes delta sites, URA3 andexpression cassette was isolated by PCR or restriction digest andtransformed into M2125. Some enzymes were transformed individually andothers were transformed in combinations. When more than one gene wastransformed, different DNA fragments were mixed in equal ratio (totalDNA amount the same as for single genes, about 1 μg). For eachtransformation about 100 colonies were picked (one 96 wp) andpre-screened by specific assays (for example, xylan assay for xylanasesintegrated, starch assay for alpha-amylases). Consequently several ofthe most active transformants were assayed by corn flour assay andscreened for increased sugars release. For each assay, transformantswere grown in YPD for 3 days and supe was assayed. The example ofsecondary corn flour assay is shown on FIG. 74. FIG. 74 shows that manytransformants demonstrated activity above parental M2111 strain. Thetransformations screened in this experiment are described in the Table27.

Transformers that released the most sugars in corn flour assay(highlighted in FIG. 74) were selected for screening by fermentation.First strains were pre-screened by minivial fermentation assay on twosubstrates: homemade corn mash and raw corn flour. Homemade mash andcorn flour were picked as screening substrates because they allow betterdiscrimination between different strains (tougher substrate), whileindustrial corn mash is too digestible to allow noticing the smalldifferences between strains. Each substrate generated different groupsof the best performers. The only strains that seemed to perform well onboth substrates were strains with AE7 (Debaryomyces occidentalisalpha-amylase) integrated. The next step of screening was scaled up toshake flasks and also was done on the same two substrates, but differentsets of strains were used for each substrate depending on performance inminivials assays. The results of shake flask screening experiments areshown on FIGS. 75 and 76. FIGS. 75 and 76 show that several differentsaccharolytic genes and their combinations had positive effect onethanol titer. Confirming the minivials assay results, AE7 had positiveeffect on both substrates.

Remaking the M2111 strain was attempted in order to increase AE9production. It was noticed that there is a significant activityvariation between transformants even when obtained with directedintegration. Therefore, screening more transformants usually yieldsstrains with higher expression level. Only 84 transformants werescreened when M2111 was selected. In order to increase AE9 expressionlevel, M139 was transformed with the same AE9 expression construct aswas used for making the M2111 strain. The expression construct wasintegrated into FCY locus and FCY was used as negative selection. About1000 transformants were screened for starch activity. Severaltransformants demonstrated activity higher than M2111. Several of themost active on starch transformants were screened by minivialsfermentation assay on homemade mash and raw corn flour. Sometransformants had higher EtOH yield compared to M2111, on raw cornflour. In the follow up experiment, several best strains were screenedin shake flask fermentation on the same two substrates (FIG. 77). Thisexperiment confirmed that strains with higher activity on starch reachhigher ethanol titers on corn flour. On homemade mash there is nosignificant difference comparing to M2111. The performance difference onflour could be due to higher secretion level of AE9. To test thishypothesis, several of the best strains were inoculated and grown in YPDfor 3 days. AE9 was measured by HPLC. The protein data was plotted inFIG. 77 together with EtOH data. The correlation between AE9 levelproduction and EtOH yield was found for corn flour fermentation andthere is no such correlation for homemade mash. This data indicate thaton corn flour the strains are still GA limited, while on homemade mashthey are not.

The best performing strains that came out of screening on homemade mashand raw corn flour were also tested on industrial corn mash (FIGS. 78and 79) which is the most commercially relevant substrate for thisapplication (used in majority of commercial corn ethanol facilities).The best strains from that screen are summarized in Table 34.

TABLE 27 Transformations ID (T) for corn flour activity assay data fromFIG. 74. S. cerevisiae promoter used with each gene shown inparentheses. Transformation# Genes transformed 1 AE8 (TEF2p) 2 AE9(ADH1p) 3 AE10 (FBA1p) 4 AE7 (ENO1p) 5 AE1 (ADH1p) 6 AE1 (TEF2p) 7 AE8 +SBD (ENO1p) 8 BC60 (ADH1p) 9 ARA (ENO1p) 10 BC60 (ADH1p) + ABF (ENO1p)11 BC60 (ADH1p) + AXE (ENO1p) 12 BC60 (ADH1p) + FAE1 (PFK2p) 13 BC60(ADH1p) + FAE2 (PYK1p) 14 BC60 (ADH1p) + FAE1 (ENO1p) 15 SE32 (ENO1p) 16SE33 (ENO1p) 17 SE34 (ENO1p) 18 SE35 (ENO1p) 19 SE47 (ENO1p) 20 SE48(ENO1p) 21 SE35 (ENO1p) + AE8 (TEF2p) 22 SE35 (ENO1p) + AE10 (FBA1p) 23SE35 (ENO1p) + AE7 (ENO1p) 24 SE35 (ENO1p) + BC60 (ADH1p) + ARA (ENO1p)25 SE32 (ENO1p) + ABF (ENO1p) + AXE (ENO1p) 26 SE34 (ENO1p) + ABF(ENO1p) + AXE (ENO1p) 27 SE35 (ENO1p) + AE8 (TEF2p) + AE7 (ENO1p) + AE10(FBA1p) 28 AE8 (TEF2p) + AE10 (FBA1p) + AE1 (ADH1p) 29 Empty vectorcontrol 30 No DNA control 55 BC60 (ADH1p) + ARA (ENO1p)

TABLE 28 Strains expressing additional to AE9 saccharolytic enzymesselected for screening on corn mash in shake flasks (FIG. 75). Strain #ID Strain description 1 T4-8 M2111 + AE7 2 T4-1 M2111 + AE7 3 T5-88M2111 + AE1 4 b M2111 + AE1 + AE8 + AE10 5 d M2111 + AE1 + AE8 + AE10 6i M2111 + AE1 + AE8 + AE10 7 o M2111 + AE1 + AE8 + AE10 8 r M2111 +AE1 + AE8 + AE10 9 M2111 Control 10 M139 Control

TABLE 29 Strains expressing additional to AE9 saccharolytic enzymesselected for screening on corn flour in shake flasks (FIG. 76). Strain #ID Strain description 1 T2-6 M2111 + AE9 2 T11-32 M2111 + BC60 + SE66 3T18-11 M2111 + SE35 4 T5-88 M2111 + AE1 5 T4-8 M2111 + AE7 6 b M2111 +AE1 + AE8 + AE10 7 i M2111 + AE1 + AE8 + AE10 8 p M2111 + AE1 + AE8 +AE10 9 M2111 Control 10 M139 Control

TABLE 30 Strains expressing AE9 selected for screening on homemade cornmash in shake flasks (FIG. 77, top). Strain # ID Strain description 1P4-19 M139 + AE9 2 P8-60 M139 + AE9 3 P11-67 M139 + AE9 4 P12-65 M139 +AE9 5 P12-17 M139 + AE9 6 M2111 Control 7 M139 Control

TABLE 31 Strains expressing AE9 selected for screening on corn flour inshake flasks (FIG. 77, bottom). Strain Strain # ID description 1 P2-9M139 + AE9 2 P11-20 M139 + AE9 3 P12-84 M139 + AE9 4 P12-65 M139 + AE9 5P12-17 M139 + AE9 6 M2111 Control 7 M139 Control

TABLE 32 The best strains from shake flask screening experiments onhomemade mash and corn flour (FIGS. 75-77) selected for screening onindustrial corn mash in shake flasks (FIG. 78). Strain # ID Straindescription MXXXX 1 T2-6 M2111 + AE9 M2327 2 T4-1 M2111 + AE7 M2328 3T4-8 M2111 + AE7 M2329 4 P11-67 M139 + AE9 (1000 colonies screen) M23305 P12-65 M139 + AE9 (1000 colonies screen) M2331 6 P12-84 M139 + AE9(1000 colonies screen) M2332 7 T2-25 M2111 + AE3 + AE5 + AE7 (D.o.genes) M2333 8 T2-40 M2111 + AE3 + AE5 + AE7 (D.o. genes) M2334 9 T2-64M2111 + AE3 + AE5 + AE7 (D.o. genes) M2335 10 T3-32 M2111 + AE3 + AE5 +AE7 (D.o. genes) M2336 11 M2111 Control 12 ER Control 13 M139 Control

TABLE 33 The best strains from shake flask screening experiments onhomemade mash and corn flour (FIGS. 75-77) selected for screening onindustrial corn mash in shake flasks (FIG. 79). Strain # ID Straindescription MXXXX 1 T5-88 M2111 + AE1 M2337 2 T18-11 M2111 + SE35 M23383 b M2111 + AE1 + AE8 + AE10 (S.f. genes) M2339 4 d M2111 + AE1 + AE8 +AE10 (S.f. genes) M2340 5 p M2111 + AE1 + AE8 + AE10 (S.f. genes) M23416 T6-88 M2111 + SE32 + ABF M2342 7 T11-12 M2111 + SE34 + AXE M2343 8T11-45 M2111 + SE34 + AXE M2344 9 T12-81 M2111 + SE34 + FAE1 M2345 10M2111 Control 11 ER Control 12 M139 Control

TABLE 34 Strains selected as best performers on industrial corn mash(FIGS. 78 and 79). Strain ID Strain description T11-45 M2125 + SE34 +SE66 T2-40 M2125 + AE3 + AE5 + AE7 T4-8 M2125 + AE7 P12-84 M139 + AE9

Example 33: Stability of Strains Built by Directed and RandomIntegration

Stability of the M2111 strain built by directed integration was tested.M2111 demonstrated remarkable stability. There was no decrease inactivity up to 99 generations in non-selective YPD media (FIG. 80, top).In order to test if random integration strains have sufficient stabilityfor use in industrial fermentation, two of the best performing strainsfrom homemade mash and corn flour shake flask fermentation experiment,T4-1 (M2125+AE7) and T2-6 (M2125+AE9) (FIGS. 75 and 76) were subjectedto the same stability test as M2111 (FIG. 80, bottom). FIG. 80 showsthat even though these tested random strains do not have the same levelof stability as directed M2111, they lost very little activitythroughout propagation on YPD. There is no loss in activity for upto9-10 generations. Only 10% is lost at about 50 generations, and 20% atabout 99 generations. The pattern of activity decrease was very similarfor two different random strains. During industrial yeast preparationcells go through about 28 generations (volume increased 300000000times). In propagation stage cells go though about 4 generations and 4generations during fermentation. Thus, the total number of generationsis about 36. Therefore, no significant activity will be lost during allstages of industrial application, considering that only 10% is lost atabout 50 generations.

Example 34: Integration Strategies for Directed Strains ConstructionExpressing Multiple Enzymes

FIG. 81 demonstrates one site integration strategy (top) and multiplesites strategy (middle) that could be used to construct strainsexpressing multiple enzymes. In one site strategy, negative markersalternate in each transformation round and all expression cassettesintegrated into the same locus next to each other. In multiple sitesstrategy, positive and negative markers alternate with each other and ineach round of transformation the expression cassette can be integratedinto any site on chromosome.

Example 35: Expression of Several Cellulolytic Enzymes in a Single YeastStrain for Hydrolysis of Wood

From the data generated by mixing several cellulases in assays in eithercrude or purified form, it was determined that a strain producingmultiple cellulolytic activities would increase the ability of theexpressing strain to hydrolyze lignocellulose. To test this idea,strains of S. cerevisiae that expressed up to 7 enzymes simultaneouslywere created. Briefly, a robust, xylose utilizing strain, M1577, wasfirst engineered to make high levels of the C. lucknowense CBH2.

Two transformations were carried out in series to generate this strain.In the first step, plasmid pMU2115 was digested with NotI to create anintegration cassette that targets a CBH2 expression and zeocin selectioncassette to the rDNA loci. Colonies from this transformation wereselected for on yeast extract (10 g/L), peptone (20 g/L), and xylose (20g/L) containing agar with zeocin (YPX+zeo), picked, and screened forenzyme activity in an avicel assay protocol. Once the best transformantfrom those screened was identified, this transformant was transformedagain with 2 additional constructs for CBH2 expression. One of these,pMU2143 (digested with NotI) targets a CBH2 expression construct and thekanamycin resistance marker to repeated tau1 genomic loci in S.cerevisiae. The other plasmid, pMU2142 (also digested with NotI) targetsa CBH2 expression construct and the hygromycin resistance marker torepeated tyB genomic loci. Following this second transformation andselection on YPX agar plates with zeocin, hygromycin, and G418 present,colonies were again screened using the avicel assay method describedbelow. The strain with the highest CBH2 production was stored and namedM1873. M1873 is capable of producing ˜150 mg/L of CBH2 in shake flaskfermentations as measured by a HPLC assay.

M1873 was subsequently transformed with PCR cassettes that wereassembled by yeast via homologous recombination to create a cassettethat allows for co-expression of four cellulases (endoglucanases) at theS. cerevisiae FCY1 locus. These four endoglucanses were EG1 fromAspergillus fumigatus, EG2 from Trichoderma reesei, EG3 from Neosartoryafischeri, and Cel9A from Thermobifida fusca, all under control ofdifferent promoters and terminators from S. cerevisiae (ENO1promoter/PYK1 terminator, PMA1 promoter/ENO1 terminator, TPI1promoter/FBA1 terminator, and PDC1 promoter/ENO2 terminator). Table 35lists the primers and templates used to generate the proper fragmentsfor assembly. Table 37 lists all the primer sequences and the plasmidsequences are listed below as well. After transformation, strains wereselected for resistant to 5-fluorocytosine, which is toxic to cells thathave an intact FCY1 locus. In addition, strains were checked for theirresistance to Clonat, and checked by PCR (X10821/X10824) for an in tactFCY1 locus. Strains showing Clonat resistance and no native FCY1 locuswere screened for activity using the CMC activity assay, and the PHWassay. The strain producing the most glucose from PHW was stored andcalled M2217. The retention of CBH2 production was confirmed by the HPLCassay.

After M2217 was built, a final transformation was used to generatestrains that also expressed the Talaromyces emersonii CBH1 fused withthe CBD from Humicola grisea (pMU1392). This was carried out in the sameway as described above, only with a different set of PCR products. Inaddition, two pieces for the gene assembly were derived from a digestionof a plasmid, rather than as a PCR product. Table 36 lists the fragmentsused. Two copies of an expression cassette for a gene encoding a fusionprotein between the T. emersonii CBH1 and the Humicola grisea CBD (fromthe H. grisea CBH1) were placed facing each other with integrationflanks specific to the δ sites of the Ty1 transposon (FIG. 82).Following transformation cells were plated to media containing 6.7 g/LYeast Nitrogen Base and 20 g/L Cellobiose as the sugar source. Thismedia allows for selection of transformants based on selection forexpression and secretion of the S. fibuligera BGLI. Transformants werethen screened for activity in the PHW assay and the top candidates werestored and given the numbers M2230, M2231, and M2232.

After this set of strains had been built a final comparison was carriedout using the PHW assay. Briefly, the set of strains was grown upaerobically in YPD media for 2 days in 48 well plates. The supernatantsfrom these cultures were added to PHW (4% total solids finalconcentration), along with a small amount (2 mg/g) of cellulase enzymefrom Trichoderma reesei supplied by AB Enzymes and buffer. The amount ofglucose released from the PHW was followed over time by HPLC. The datafrom this comparison can be found in FIG. 83. M1873, producing only theC. lucknowense CBH2 provides a large increase in activity relative tothe control strain M1577 in this test—an approximate 176% increase inglucose release. The addition of set of four endoglucanases, providesanother increase relative to M1873 of 18%, and the addition of CBH1 andBGL provide another 28% increase above that. Overall, strains producing7 cellulolytic enzymes increase hydrolysis over the negative controlstrain by >3 fold over the control strain, and by >50% relative to astrain producing only a single enzyme.

A set of strains from those described above was subsequently tested forits ability to impact the amount of ethanol produced from pretreatedhardwood. FIG. 84 presents data from simultaneous saccharification andfermentation (SSF) reactions containing a small amount of externallyadded cellulase enzyme. SSF conditions were as follows: final solidsloading was 18% (w/w) of substrate MS887 (an insoluble substrate derivedfrom pretreating hardwood with water), 2 mg AB Enzyme cellulasepreparation/g total solids, 10% v/v inoculum, 35° C., pH 5.5 controlledwith 5 g/L CaCO₃. The medium used was Corn Steep Liquor (CSL, 12 g/L)and diammonium phosphate (DAP, 0.5 g/L). Reactions were carried out insealed plastic centrifuge bottles, fitted with vents and mixed via largestir bars, by combining all the above ingredients in a 100 gram finalmass batch culture, mixing at 225 rpm on a shaker, and sampling over 160hours. M1873 and M2232 both were able to produce more ethanol underthese conditions than non-cellulolytic M1577. M1873 could increase yieldby 15% and 33% relative to M1577 on unwashed and alkaline washedpretreated hardwood respectively. M2232 could produce 20% and 43% moreethanol than M1577 on these two substrates. The ability of M2232 toproduce more ethanol that M1873 demonstrates the utility of expressingthe package of 7 enzymes simultaneously in a single strain.

TABLE 35 PCR fragments used to assemble EG expression islands in S.cerevisiae. Piece ID No. Description Primers Template 1 FCY f1X11631/X12837 gDNA 2 EG1 X12838/X12822 pMU1821 3 EG2 X12823/X12824pMU1479 4 EG3 X12825/X12826 pMU1958 5 Cel9A X12827/X12828 pMU1975 6Clonat Marker X12829/X12841 pMU227 7 FCY f2 X12842/X11634 gDNA

TABLE 36 PCR fragments used to assemble CBH1 expression islands in S.cerevisiae. Piece ID No. Description Primers Template 1 Delta f1X12427/X13008 gDNA 2 Eno1p- NA Digest of pMU1392 TeCBH1 + HgCBD withSmaI and AscI 3 CYC term 1 X13009/X13010 pMU2142 4 AgTef termX13011/X13012 pMU183 5 SfBGL NA pMU1260 digest with PacI/AscI 6 AgTefprom X13013/X13014 pMU183 7 CYC term 2 X13009/X13015 pMU2142 8 Delta f2X13016/X12434 gDNA

TABLE 37 Primers used in the construction of these strains Primer SEQ IDName Sequence (5′-3′) Description NO X10821 AAGAGGGTGGTGTTCCTATTGGCGGATFCY check for 526 GTCTTATCAATAACAAAGACGGAAGT GTTCTC X10824TTTTGAAATTAACGTTCTCACCGACAA FCY check rev 527 CACAGCGTGGAATACCATACATGATGATGGCA X11631 TTGCCAAAGTGGATTCTCCTACTCAAG FCY f1 for 528 CTTTGCAAACATX12837 GAAGCTCGGATCAGTAGATAACCCGC FCY f1 rev 529CTAGAAGACTAGTAGCTATGAAATTTT TAACTC X12838 GAGAGCCAGCTTAAAGAGTTAAAAATEG1 for 530 TTCATAGCTACTAGTCTTCTAGGCGGG TTATC X12822GTTTTTTCCCCGTCAGCGATGGTGACG EG1 rev 531 TAAACGACTAGATTTAGGACACTAATTGAATC X12823 AAAAAATGACGCGGGCAGATTCAATT EG2 for 532AGTGTCCTAAATCTAGTCGTTTACGTC ACCATC X12824 GATGGGTTCCTAGATATAATCTCGAAGEG2 rev 533 GGAATAAGTAGGCAAAGAGGTTTAGA CATTG X12825GTTCTAAGCTCAATGAAGAGCCAATGT EG3 for 534 CTAAACCTCTTTGCCTACTTATTCCCTTCGAG X12826 GTTTATTACATGAAGAAGAAGTTAGTT EG3 rev 535TCTGCCTTGCTTGCTAGAGAATAAATT CAAG X12827 GTTCAACATCATCTTTTAACTTGAATTTCe19A for 536 ATTCTCTAGCAAGCAAGGCAGAAACT AAC X12828CGGGTGACCCGGCGGGGACGAGGCAA Ce19A rev 537 GCTAAACAGATCTCAAACAACTTAAAATCAGTC X12829 GGCATATCAAGACCCTGCCTGGACTGA Clonat for 538TTTTAAGTTGTTTGAGATCTGTTTAGCT TGCC X12841 ATATAAAATTAAATACGTAAATACAGCClonat rev 539 GTGCTGCGTGCTATTAAGGGTTCTCGA GAGC X12842CCAGTGTCGAAAACGAGCTCTCGAGA FCY f2 for 540 ACCCTTAATAGCACGCAGCACGCTGTATTTACG X11634 TAGCCCTTGGTTGAGCTTGAGCGACGT FCY f2 rev 541 TGAGGT X12427GGCCGCTGTTGGAATAAAAATCC Delta f1 for 542 X13008CTCGGATCAGTAGATAACCCGCCTAGA Delta f1 rev 543 AGACTAGTGGATCGATCCCCGGGATGTTTATATTCATTGATCCTATTACATTATC AATCC X13009 ATCTGTACCAAGTTGAACGACTGGTACCYC term 1 for 544 TCTCAATGTTTATAAGGCGCGCCACAG GCCCCTTTTCCTTTG X13010CCGCCATCCAGTGTCGAAAACGAGCTC CYC term 1 rev 545 GTCGACAACTAAACTGGAATGTGX13011 CCTCACATTCCAGTTTAGTTGTCGACG AgTef term for 546AGC TCGTTTTCGACACTGGATGG X13012 GCTGTTAATGATATCAAGACATCTGTCAgTef term rev 547 CTGTTTACTATTTGAGGCGCGCCTCAG TACTGACAATAAAAAGATTCTTGX13013 GCGACGCCGGCGAGGAGGGAGGTGAA AgTef prom for 548GGAGACATTTTGTTTTTAATTAAGGTT GTTTATGTTCGGATGTGATG X13014TTGTTGTTCCCTCACATTCCAGTTTAGT AgTef prom rev 549 TGTCGACAGCTTGCCTTGTCCCX13015 GGTGACCCGGCGGGGACAAGGCAAGC CYC term 2 rev 550TGTCGACAACTAAACTGGAATGTG X13016 GCTCAATTAGTGGACGTTATCAGG Delta f2 for551 X12434 CCGCGGTGAGATATATGTGGGTA Delta f2 rev 552

Example 36: Expression of Accessory Enzymes in Yeast

For the proteins described below, various enzymes were expressed inyeast in their native form as well as with the addition of a cleavableHis tag for the purposes of increased ease of purification. Proteinswere assayed with and without the His tag to determine if the taginfluenced the activity or banding pattern of the protein. If deemednecessary, tags can be removed in subsequent enzyme evaluation assaysafter cleavage with enterokinase and re-purification. Genes were PCRamplified or codon optimized and synthesized and cloned into vectorpMU1531 that had been digested with Pac1 and Asc1. A C-terminalenterokinase site expressed as amino acids DDDDK, linker expressed asamino acids GGSPPS and 6×His tag expressed as amino acids HHHHHH wereadded by yeast via homologous recombination, and constructs weresequenced to confirm the tag sequence was intact and the gene and tagwere in-frame.

Colonies from transformations were grown in indicated media for 48-72hours. Cultures supernatants were filtered through a 2 um PE filter andconcentrated approximately 20-fold using 10,000 molecular weight cut offfilters. Protein quality was screened via SDS-PAGE electrophoresis undernon-reducing conditions.

Expression of Alpha-Glucuronidase in Yeast

Pichia stipitis alpha-glucuronidase, GH67 (NCBI#ABN67901) was expressedin yeast (FIG. 85). Alpha-glucuronidase is predicted to be approximately111 kDa (untagged) and 113 (C-terminal His tagged), and is seen as aband between 100 and 150 kDa in FIG. 85. Most GH67 alpha-glucuronidasescharacterized to date liberate MeGlcA residues linked to terminalxylopyranosyl residues. The protein described here liberates MeGlcAresidues linked to terminal and internal xylopyranosyl residues (Ryabovaet al, FEBS Letters 583:1457-1462, (2009)).

Expression of Xyloglucanases in Yeast

Several xyloglucanases (Table 38) were functionally expressed in S.cerevisiae (FIGS. 86-87). The strain expressing Aspergillus niger XGLproduced the most activity; however, His tag addition had a negativeeffect on activity (about 50% less activity at 1 hour).

Secreted xyloglucanases were also characterized by Silver stainedSDS-PAGE and Westen blot analysis (FIG. 88). On SDS-PAGE a large clearband was visible for Aspergillus niger xg11 (˜150 kDa); no band forAspergillus aculeatus xg11; and a discrete band for Neosartorya fischerixg1 (˜130 kDa). His tag versions of the proteins showed apparently lesssecreted protein. The Western blot analysis showed that the signals forthe Aspergillus niger xglHis tag was strong; for Neosartorya fischerixglHis tag was poor, and A.c.xgl-His tag was not visible. Trichodermareesei xgl+/−His tag was not examined due to undetectable activity inthe AZCL xyloglucan assay.

TABLE 38 Xyloglucanases expressed in Saccharomyces cerevisiae AccessionUntagged Tagged Activity: Enzyme: Organism: number Plasmid size sizexyloglucanase GH74A Trichoderma AAP57752 pMU2088 87.0 kDa 88.9 kDa(EGL6) reesei GH74A Aspergillus AAK77227 pMU2856 90.3 kDa 92.2 kDa(EGL6) niger GH74A Aspergillus BAA29031 pMU2857 89.7 kDa 91.6 kDa (EGL6)aculeatus GH74A Neosartorya XP_001261776 pMU2858 89.3 kDa 91.2 kDa(EGL6) fischeri XG*Expression of Esterases in Yeast

Several esterases (Table 39) were functionally expressed in S.cerevisiae. The expression was characterized by SDS-PAGE (FIG. 89) andactivity assay (FIG. 90). SDS-PAGE analysis demonstrated thatAspergillus niger FAEA (pMU1880) showed a prominent band at ˜36 kDa,Chaetomium globosum FAEB (pMU1882) showed multiple visible bands, and nobands were noted for Aspergillus terreus FAEA (pMU1884). Prominent bandswere visible for Chaetomium globosum CIP2 (pMU2095+/−C His tag) andTrichoderma reesei CIP2 (pMU2097) glucuronyl esterases.1-Napthtyl-acetate was used to assay ferulic acid esterases (FIG. 90),but this substrate did not work well for the glucuronoyl esterases.Glucuronoyl esterases were not tested further for activity. Aspergillusniger FAEA (pMU1880) exhibited the best activity on this substratefollowed by Chaetomium globosum FAEB (pMU1882).

TABLE 39 Esterases expressed in Saccharomyces cerevisiae AccessionUntagged Tagged Activity: Enzyme: Organism: number Plasmid size sizeferulic CE1 Aspergillus XP_001393337 pMU1880 30.5 kDa 32.4 kDa acid/(FAEA) niger cinnamoyl CE1 Aspergillus XP_001211092 pMU1884 35.5 kDa37.4 kDa esterase (FAEA) terreus CE1 Talaromyces EED17739 pMU1881 37.5kDa 39.4 kDa (FAEB) stipitatus CE1 Chaetomium XP_001228412 pMU1882 36.7kDa 38.6 kDa (FAEB) globosum glucuronyl CIP2 Trichoderma AAP57749pMU2097 48.2 kDa 50.1 kDa esterase reesei CIP2 Chaetomium XP_001226041pMU2095 49.8 kDa 51.7 kDa globosumExpression of α-Galactosidases in Yeast

Several alpha-galactosidases (Table 40) were functionally expressed inyeast (FIGS. 91-93). All AGL1 and 2 expressing strains exhibitedsecreted activity (FIG. 91), but the His tag had a negative impact onactivity (decreased by about 50%). AGL3 strains were not available fortesting at the time these experiments were conducted.

Alpha-galactosidases were also analyzed by Western blot (FIG. 92) andsilver stain (FIG. 93). Trichoderma reesei AGL3 sample had one prominentband at approximately 50-70 kDa by Western blot. On SDS-PAGE visible(smeared) bands (over 100 kDa) are noted for Trichoderma reesei agl1 andTalaromyces emersonii agl1 (predicted sizes: 48.5 & 49.4 kDa); discreetband of ˜80 kDA noted for Trichoderma reesei agl2 (predicted size: 82kDa), but was poorly expressed (not shown).

TABLE 40 Alpha-galactosidases expressed in Saccharomyces cerevisiaeAccession Untagged Tagged Activity: Enzyme: Organism: number Plasmidsize size alpha- GH27 Trichoderma CAA93244 pMU2859 48.4 kDa 50.3 kDagalactosidase (AGL I) reesei GH27 Talaromyces EU106878 pMU2860 49.3 kDa51.2 kDa (AGL I) emersonii GH27 Trichoderma Z69254 pMU2861 82.0 kDa 83.9kDa (AGL II) reesei GH27 Trichoderma CAA93246 pMU2697 67.0 kDa 68.9 kDa(AGL reesei III)

Example 37: Enzymatic Conversion of Pretreated Mixed Hardwoods

To assess the effect of various enzymes on pretreated mixed hardwoods(PHW), an assay was conducted with 2% solids, pH 5.0 and 38° C.Yeast-produced and purified enzymes were assessed in the assay eitherwith or without additional commercial enzymes. The activity of the mixwith yeast-produced enzymes evaluated by the release of sugars,predominantly glucose due to the nature of the pretreatment, by HPLCusing a BioRad 87H column. The data below shows the results of some ofthose mixing experiments. FIG. 94 shows that the addition of CBH2, BGL,EG1, EG2 and EG3 improves hydrolysis of the substrate above what thecommercial enzyme mix can do with just the addition of CBH2 and BGL.Therefore, yeast-made EG1, EG2 and EG3 provide benefits in hydrolyzingPHW. FIG. 95 shows that further addition of yeast-produced and purifiedxylanase, xylosidase and AXE improved hydrolysis of the PHW above whatwas seen with either just the commercial enzyme mix or the commercialmix with CBH2 added. This further suggests the benefits of the accessoryenzymes described above.

FIG. 96 shows that the addition of these enzymes in combinationcontinues to show improvement over the addition of just one of theaccessory enzymes.

Example 38: Enzymatic Conversion of Paper Sludge

The information above was done on PHW in the presence of commercialenzymes. The following data shows the effectiveness of the purified,yeast-produced enzymes to hydrolyze paper sludge without any additionalenzymes added in both a 2%, pH 5.0, 38° C. hydrolysis assay as well asan SSF. These results are compared to the same assay or fermentationwith the addition of commercial enzymes.

These data in FIGS. 97 and 98 show that the combination of CBH1, CBH2,BGL, EG1, EG2, EG4, EG5, xylanase and xylosidase hydrolyze moresubstrate when combined together than when assayed alone. This wasfurther confirmed in fermentation (FIG. 99). The purified enzymes wereanalyzed by SSF on two different types of industrial paper sludge. Bothpaper sludge substrates were washed with 1M citric acid. The SSFs werecarried out under the following conditions: 2% total solids, 1.1 g/L drycell weight M2108, 15 mg/mL Tetracycline, YP media, pH 5.0, 35° C. and220 rpm. A selected cocktail of yeast made enzymes was dosed at 4.1 mg/gTS and compared to a dose response of AB Whole Broth ranging from 0-6.1mg/g TS. The purified enzyme cocktail is specified in Table 41 and theresults are shown in FIG. 99. Based on data shown on FIG. 99, the yeastmade enzyme dose is equivalent to a dose of approximately 3 mg/g TS ABWhole Broth commercial enzymes mix on both substrates. These datasupport the claim that the combination of the yeast-produced, purifiedenzymes can hydrolyze industrially relevant substrates such as papersludge without any additional commercial enzymes. Generated by yeastmade enzymes sugars are successfully converted by yeast to ethanol inSSF process.

TABLE 41 Yeast made enzyme cocktail used in paper sludge SSF. dose (mg/gEnzyme TS) TeCBH1 with Hg CBD 2.25 Cl CBH2 0.7 Sf BGL 0.1 Af EG1 0.35 HjEG2 0.15 Tt EG4 0.05 Cl EG5 0.05 EG6 0.05 An Xyn 0.2 Ptr Xld 0.2 Total4.1

TABLE 42 Summary of the best yeast expressed cellulases, hemicellulasesand accessory enzymes. Highlighted yellow - key enzymes for woodconversion; Yellow + Green - key enzymes for paper sludge conversion(based on data shown in FIGS. 94-99). Type of Cazy family/Well-Expressed Accession Activity enzyme type Candidates Numberexoglucanase GH7A (CBH1) T. emersonii See underlined CBH1 + HgCBD orf inpMU1392 GH6A (CBH2) C. lucknowense See patent (CBH2) applicationWO/2010/060056 endoglucanase GH7B (EG1) A. fumigatus EG1 XP_747897 GH5A(EG2) T. reesei EG2 See patent application WO/2010/060056 GH12A (EG3) N.fischeri EG3 XP_001261563 GH61A (EG4) T. terrestris EG4 ACE10231 GH45A(EG5) C. lucknowense ACH15008 EG5 GH6 (EG6) N. crassa EG6 XP_957415 GH5(bact.) C. cellulolyticum YP_002505438.1 Cel5A GH? (bact.) B. subtilisEGLS CAB13696.2 GH9 (bact.) T. fusca Cel9A YP_290232 GH8 (bact.) C.cellulolyticum AAA73867.1 Cel8c xyloglucanase GH74A A. niger XG AAK77227(EGL6) β- BGLI S. fibuligera BGLI See patent glucosidase applicationWO/2010/060056 xylanase GH11 (XYN2) T. reesei xyn2 ABK59833 GH10 A.niger xyn10 CAA03655.1 β-xylosidase GH3 A. niger Xld3 XP_001389416 GH43(BXL1) Pyrenophora XP_001940956 tritici-repentis BXL beta- GH5 (MAN1) A.aculeatus MAN5 AAA67426 mannase beta- GH26 C. phytofermentensYP_001559376.1 mannosidase mannosidase acetylxylane CE1 (AXE) N.fischerii AXE1 XP_001262186 sterase arabinofuran GH54 (ABF1) A. nigerABFB AAA93264 osidase ferulic CE1 (FAEA) A. niger FAEA XP_001393337acid/cinnamoyl CE1 (FAEB) T. stipitatus FAEB EED17739 esterase A- GH67Pichia stipitis ABN67901 glucuronidase

Example 39: Strain Identification and Activities for Strains Tested on30% TS Corn Flour

Supernatants were assayed on the supernatant remaining at the end of acorn mash fermentation to determine if any of these enzymes couldfurther hydrolyze the soluble oligomers. Cell supernatants of strainsengineered with α-glucosidase activity released glucose from solubleoligomers remaining at the end of a corn mash fermentation. The increaseobserved was higher than cell supernatant from the background strain(M749). All samples contained a blanket dose of commercial glucoamylase.

The control M0139 with 0.3 AGU/g TS GA reaches 121 g/L ethanol withpotential ethanol of 127 g/L. M2111 is a bit higher with respect to bothethanol produced and potential ethanol, showing a CBP effect. There area handful of strains that have potential ethanol of over 128 g/L, withT2-6 at 133 g/L. T2-6 (AE9) reached the highest ethanol titers as well,125 g/L. T11-32 (BC60, AXE) also has potential ethanol over 130 g/L. Allof these strains show a CBP effect over the control strain.

TABLE 43 Groups of enzymes used in evaluation of pretreated wet cakewith the addition of supernatants Protein Group Name CBH1 Big 4 Big 6CBH2 EG2 BGL Xyl Big 2 Xld

M0139 is the control strain and has no enzymatic activities. Eachyeast-made purified enzyme was added to the control strain and a smallbenefit is seen. When added together, as seen with the Big 4 or Big 6, alarge increase in hydrolysis is seen. The largest glucose and xyloseyields are seen with the addition of 1 mg/g TS commercial Pectinase(Multifect) to the Big 6.

Example 40: Strain Identification and Activities Expressed inSupernatant that were Evaluated on Pretreated Wet Cake (ELN Afoster2Corn-074

Corn wet cake that was pretreated by autohydrolysis in the steam gun(30% TS, 160° C., 20 minutes) was used to evaluate the effect onhydrolysis when yeast-made purified enzymes are used in the presence ofa mixture of commercial enzymes. The mixture of commercial enzymes(referred to as MM) used was 0.9 mg/g TS AB Whole Broth, 0.1 mg/g TSMultifect Pectinase and 0.1 mg/g TS Spirizyme GA. Purified CBH1 wasadded at a concentration of 1 mg/g TS where all other purified enzymeswere added at 0.25 mg/g TS. These enzymes were added to 2% TS pretreatedwet cake (PWC), 75 mM Na citrate buffer pH 5.0, 0.01% Na Azide to atotal volume of 4 mLs in a 24 well plate. The hydrolysis was incubatedat 35° C., 220 rpm. The 48 hour results are shown in FIG. 102.

The glucose released with just the commercial enzyme mix “MM” is 2.8g/L. When purified yeast made enzymes are then loaded in addition to“MM,” an increasing trend in hydrolysis is observed. When all of thepurified enzymes are added without “MM,” (shown in the last bar on theright side of the graph), glucose release is still observed. Theaddition of purified enzymes with or without commercial enzymes showshydrolysis. Corn coarse fiber (similar to wet cake but with the proteinremoved) was pretreated in the steam gun at 190° C. for 10 minutes withwater where another condition used 1% sulfuric acid for thepretreatment. These two substrates were evaluated in the presence of acommercial enzyme mixture with the addition of purified yeast madeenzymes, similar to the previous experiment. The purpose of thisparticular assay was to determine the best ratio of purified CBH1 andCBH2 in the presence of 1 mg/g TS commercial enzyme mixture of C-tec:H-tec: Multifect Pectinase at ratios of 30%: 45%: 25% with 0.5 U/gTSDepol FAE. The various mixtures used are specified in Table 44 and theresults are shown in FIG. 103.

TABLE 44 Mixtures of purified enzymes used to determine the optimalratio of CBH1 to CBH2 on pretreated corn coarse fiber. The commercialmixture of C-tec:H-tec:Multifect Pectinase at ratios of 30%:45%:25%with0.5 U/gTS Depol FAE was dosed at 1 mg/g TS to each sample mg/g TSug/mL Mix 1 Mix 2 Mix 3 Mix 4 Mix 5 Mix 6 Mix 7 Mix 8 Mix 9 CBH1 2.45 32.25 1.5 0.75 0 4 2.25 2.25 2.25 CBH2 1.24 0 0.75 1.5 2.25 3 0 0.75 0.750.75 BGL 0.83 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 XYN(SE34) 1.48 0 0.50.33 XLD 3.34 0 0.5 0.33 AXE 1.18 0 0.33 Comm 5 1 1 1 1 1 1 1 1 1 enzymemix Total yeast- 3.1 3.1 3.1 3.1 3.1 4.1 4.1 4.1 4.1 made enzyme

Results showed that decreasing amounts of CBH1 correlate to a decreasein glucose yields. This effect was more dramatic on the acid pretreatedcoarse fiber than on the 190° C., 10 min substrate. When 4 mg/g TS CBH1only is added, there is an equal or better yield seen than when there isCBH2 present. In short, the more CBH1, the better the glucose yields.Additions of XLD, XLN and AXE (0.33 mg/g TS each) also helped boostfinal yields a small amount over the commercial enzyme mixture.

Example 41: Methods

Yeast Strains

M0509 (NCPy102; ura-3::kanMX/ura-3::kanMX gre3::loxP/gre3::loxPTAL1+/loxP-PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 RPE1+/loxP-PTPI-RPE1TKL+/loxP-PTPI-TKL delta:: PTPI-xylA PTPI-XKS) and M0749 (NCPy102;ura-3::kanMX/ura-3::kanMX gre3::loxP/gre3::loxP TAL1+/loxP-PTPI-TAL1RKI1+/loxP-PTPI-RKI1 RPE1+/loxP-PTPI-RPE1 TKL+/loxP-PTPI-TKLdelta::PTPI-xylA PTPI-XKS fur1Δ::Nat/FUR1) strains derived from diploidwine strain NCP Y120 (obtained from University of Stellenbosch, SouthAfrica) and are described in McBride et al., WO 2010/060056, 2010. M0139(MAT a/MAT alpha) is S. cerevisiae diploid wine strain that was receivedfrom University of Stellenbosch. M1744 is derivative of M0139 withdouble URA3 knockout (markerless). Ethanol Red (ER) is commerciallyavailable diploid ethanologen strain that was obtained from LesaffreCorp.

Starch-DNS Assay

Reagents:

-   -   Dinitrosalicylic Acid Reagent Solution (DNS), 1%        (Could be stored at 4° C. for several months)    -   3,5-dinitrosalicylic acid: 10 g    -   Sodium sulfite: 0.5 g    -   Sodium hydroxide: 10 g    -   Add water to: 1 liter    -   Calibrate DNS by glucose (use glucose samples with conc.        0,1,2,3,4,5 and 6 g/l, calculate the slope [S])    -   Starch 2.2%, pH 5.0        (Prepare fresh before use; will be diluted by enzymes to 2%)    -   Dissolve 1.1 g of corn starch in 50 ml of water in a boiling        water bath    -   Add 1 ml of 3M NaAc buffer pH 5.0        Procedure:

-   1. Aliquot starch into 96w PCR plate 150 μl/well (one well for each    sample to be measured). Shake starch between refilling repeat    pipette to prevent starch settling.

-   2. Aliquot DNS into different 96w PCR plate 50 μl/well (two wells    for each sample to be measured)

-   3. Add 16.7 μl of enzyme sample (cells supernatant) into starch, mix    and immediately take 25 μl into 50 μl of DNS (control sample at t=0)

-   4. Incubate enzyme/starch samples at 35° C. for 3 h in PCR machine

-   5. Take 25 μl of enzyme/starch samples into 50 μl of DNS (t=3 h    samples)

-   6. Incubate DNS samples at 99° C. for 5 min to develop a color and    cool down at 4° C. for 5 min (use PCR machine)

-   7. Transfer 50 μl of DNS sample into 96w assay plate and measure    absorbance at 565 nm    Amylolytic activity [A] calculation (% of starch converted):    A(%)=OD ₅₆₅[t=3h]−OD ₅₆₅[t=0] g/L×100%    -   S (DNS slope) 20 g/L        Should use supernatant of cell cultures with the same growth OD.        If cells are grown differently, the activity should be        normalized by cells density.

Starch-GHK Assay

Reagents:

-   -   Hexokinase (HK) reagent        (Could be stored at −20° C. for several months)    -   Add 50 ml of water into HK reagent bottles (Sigma #G3293-50 mL)        and mix by turning up and down (usually use 6 bottles to make        stock)    -   After complete dissolving combine reagent from all bottles and        add Tris (5.45 g per 6 bottles)    -   Prepare 22 mL aliquots in 50 mL screw cap centrifuge tubes. (One        tube is sufficient to assay a 96 well microplate).    -   Store aliquots frozen    -   Calibrate each new stock by glucose standards and calculate the        slope S (with glucose conc. 2, 1, 0.5, 0.25, 0.125, 0 g/l). The        assay is linear up to 2 g/l glucose    -   Starch 2.2%, pH 5.0        (Prepare fresh before use; will be diluted by enzymes to 2%)    -   Dissolve 1.1 g of corn starch in 50 ml of water in a boiling        water bath    -   Add 1 ml of 3M NaAc buffer pH 5.0        Procedure:

1. Aliquot starch into 96w PCR plate 150 μl/well (one well for eachsample to be measured)

2. Aliquot HK reagent into 96w assay plate 200 μl/well (two wells foreach sample to be measured)

3. Add 16.7 μl of enzyme sample (cells supernatant) into starch, mix andimmediately take 10 μl and mix into 200 μl of HK reagent (control sampleat t=0). Cover with plate film and incubate HK plate at 30 C for ≥30 min

4. Incubate enzyme/starch samples at 35° C. for 3 h in PCR machine

5. Take 10 μl of enzyme/starch samples and mix with 200 μl of HK reagent(t=3 h samples). Cover with plate film and incubate HK plate at 30 C for≥30 min

6. Measure absorbance of both HK plates at 340 nm

Amylolytic activity [A] calculation (g/L glucose released):A=OD ₃₄₀[t=3h]−OD ₃₄₀[t=0] g/L

-   -   S (slope)        Should use supernatant of cell cultures with the same growth OD.        If cells are grown differently, the activity should be        normalized by cells density

Maltose Assay

Reagents:

-   -   Maltose2.2%:    -   1.1 g D-maltose    -   1 mL 3M sodium acetate buffer pH5.0    -   Bring to 50 mL with water    -   Hexokinase (HK) reagent (see Starch-GHK assay)

Procedure:

-   -   1. Aliquot 150 μL maltose solution into 96w PCR plate    -   2. Add 16.7 μL supernatant to the maltose solution    -   3. Incubate at 35 C in PCR machine for 3 h (during the last hour        get GHK reagent from freezer and allow to thaw at room        temperature—do not heat. One 50 mL tube containing 22 mL reagent        is sufficient to do one 96 well plate)    -   4. Put 10 μL of supernatant/maltose sample into a well of the        assay plate (Corning, cat#3641)    -   5. Add 200 μL of HK reagent and cover with plate film    -   6. Incubate at 35 C for ≥35 min    -   7. Measure absorbance at 340 nm    -   Amylolytic activity [A] calculation (g/L glucose):        A=OD ₃₄₀[t=3h] g/L    -   S (HK slope)        Should use supernatant of cell cultures with the same growth OD.        If cells are grown differently, the activity should be        normalized by cells density

Corn Mash Assay

Procedure:

1. Cut 1 mL tips so that there is an opening approximately 4 mm indiameter. Tips do not have to be sterile for this assay.

2. Inoculate strain to be tested in YPD. Grow with shaking for 2-3 days,35° C. to an OD₆₀₀ of approximately 8-10 (stationary phase).

3. If comparing strains, inoculate strain M0509 in YPD. Grow withshaking for 2-3 days, 35° C. to an OD₆₀₀ approximately 8-10 (stationaryphase). This will serve as a negative control in the assay.

4. Per 24-well plate, prepare substrate mix in a final volume of 100 mL:

Amount to add Final per concentration 100 mL Concentration in CM assayMaster in Master (96-well Substrate/Stock Solution Mix Mix plate)Pretreated wet corn mash 12.12 g      4%   2% (~33% solids; test on LMAand adjust the amount added accordingly) 1M Na citrate (sodium 15 mL 150mM 75 mM citrate dihydrate) pH 5.0 100X Anti-fungal/  2 mL 2X 1Xbacterial mix, Sigma #A5955 0.5% NaN3 (sodium  4 mL 0.02% 0.01% azide)in 5 mM Na citrate pH 5.0 dH20 Bring — — volume to 100 mL5. Using cut tips, add 2 mL/well of the substrate mix prepared above toa 24-well plate. Use continuous stirring with a magnetic stirrer whiledispensing the substrate. 3 replicates for each strain/condition arerecommended.6. Add 2 mL of supernatant to be assayed to each well that containssubstrate mix.7. Put 24-well reaction plate into shaker and incubate at 35° C. and 250rpm.8. Samples taken at 24 and 48 h sample by allowing the substrate in theplate to settle either by gravity or by centrifugation. Then transfer150 μL of supernatant to a centrifuge tube with a 0.2 μm filter insertor a 96-well, 0.2 μm filter plate (Fisher: Millipore part # MSGVN2250)with 7.5 μL 10% sulfuric acid added. After filtration, transfer thesample to a total recovery HPLC vial for analysis on the H-column.

Corn Fiber Assay

Procedure:

1. Cut 5 mL tips so that there is an opening approximately 4 mm indiameter. Tips do not have to be sterile for this assay.

2. Inoculate strain to be tested in YPD. Grow with shaking for 2-3 days,35° C. to an OD₆₀₀ of approximately 8-10 (stationary phase).

3. If comparing strains, inoculate strain M0509 in YPD. Grow withshaking for 2-3 days, 35° C. to an OD₆₀₀ approximately 8-10 (stationaryphase). This will serve as a negative control in the assay.

4. Per 24-well plate, prepare substrate mix in a final volume of 100 mL:

Amount to add Final Substrate/ per 100 mL Concentration concentrationStock Solution Master Mix in Master Mix in assay Washed fermentation 4.4g     4%   2% residuals (~90% solids; test on LMA and adjust the amountadded accordingly) 1M Na citrate (sodium 15 mL 150 mM 75 mM citratedihydrate) pH 4.0 0.5% NaN3 (sodium  4 mL 0.02% 0.01% azide) in 5 mM Nacitrate pH 5.0 dH20 Bring volume — — to 100 mL5. Using cut tips, add 2 mL/well of the substrate mix prepared above toa 24-well plate. Use continuous stirring with a magnetic stirrer whiledispensing the substrate. 3 replicates for each strain/condition arerecommended.6. Put 24-well reaction plate into shaker and incubate at 35° C. and 250rpm.7. Add 2 mL of supernatant to be assayed to each well that containssubstrate mix.Samples taken at 24 and 48 h sample by allowing the substrate in theplate to settle either by gravity or by centrifugation. Then transfer150 μL of supernatant to a centrifuge tube with a 0.2 μm filter insertor a 96-well, 0.2 μm filter plate (Fisher: Millipore part # MSGVN2250)with 7.5 μL 10% sulfuric acid added. After filtration, transfer thesample to a total recovery HPLC vial for analysis on the H-column.

CMC Conversion Assay

Procedure:

-   -   1. Inoculate strains to be tested in 10 mL YPD (or other media)        in 50 ml tubes and grow with shaking for 3 days    -   2. Prepare the 1.14% CMC substrate, 1.14 g CMC per 100 mL        citrate buffer (50 mM pH5.5) autoclaved for 20-25 min Agitate to        make sure all CMC is dissolved    -   3. To 44 mL of 1.14% CMC add 1 mL of 0.5% of sodium azide    -   4. Spin cells in 50 ml tubes at max speed for 10 min    -   5. Add CMC to deep well 96-well plate, 450 μL/well    -   6. Do 4 replicates for each strain    -   7. Aliquot 100 μL of DNS into 96-well PCR plate    -   8. Add 50 μL of yeast supernatant or buffer to the substrate and        mix by pipetting    -   9. Take T=0 sample: transfer 50 μL to the 96-well PCR plate        containing DNS and mix    -   10. Put the deep well plate at 35° C. 800 rpm    -   11. Heat the PCR plate at 99° C. for 5 min and cool down to        4° C. in PCR machine    -   12. Transfer 50 μL to microtiter plate    -   13. Measure absorbance at 565 nm    -   14. Take samples from reaction plate after 24 and repeat steps        6-12    -   15. Calculate % of CMC converted at time 24 hrs using formula:

$Y = {\frac{\mspace{31mu}{\left( {{{OD}\left( {T = 24} \right)} - {{OD}\left( {T = 0} \right)}} \right) \times 100\%}}{S \times A} = {\frac{\Delta\;{OD} \times 100}{0.1 \times 10} = {\Delta\;{OD} \times 100}}}$Y—% of CMC converted at 24S—DNS/glucose calibration slope that is 0.1 for DNS from May 8, 2007 at565 nmA—CMC concentration at T=0 that is 10 g/L for 1% CMCReagents:Dinitrosalicylic Acid Reagent Solution (DNS), 1%(Could be stored at 4° C. for several months)

-   -   3,5-dinitrosalicylic acid: 10 g    -   Sodium sulfite: 0.5 g    -   Sodium hydroxide: 10 g    -   Add water to: 1 liter        Calibrate DNS by glucose (use glucose samples with conc.        0,1,2,3,4,5, 6, 7, 8, 9, 10 g/l, calculate the slope [S], for        DNS from May 8, 2007 S=0.1)

Avicel Conversion Assay (High Throughput)

Procedure:

-   -   1. Inoculate strains to be tested in 600 ul YPD in deep 96-well        plate. Perform 4 repeats for each strain or 4 transformants for        each transformation. Grow with shaking for 3 days at 30° C.    -   2. Spin cells at max speed for 10 min    -   3. Prepare substrate mix:        -   Substrate mix for full 96-well plate, total volume 30 ml:        -   0.6 g Avicel (2%)        -   500 μl 3M Na Ac pH 5.0 (50 mM)        -   1.2 ml 0.5% Na Azide (0.02%)        -   30 μl BGL (Novozyme-188, Sigma)        -   Add dH2O to 30 ml    -   4. Add substrate to new deep 96-well plate, 300 μl/well. Shake        between additions; do not let the Avicel settle    -   5. Add 300 μl of yeast spined supernatant or buffer to the        substrate.    -   6. Take T=0 sample: by multichannel pipette mix the reaction mix        and transfer 100 μl to 96-well PCR plate    -   7. Put deep 96-well reaction plate into shaker at 35° C. and 800        rpm    -   8. Spin 96-well PCR plate with T=0 samples at 2000 rpm for 2 min    -   9. Aliquot 100 μl of DNS into new 96-well PCR plate    -   10. Carefully (without touching pellet) take 50 μl of super from        T=0 spined 96-well PCR plate and mix it into DNS    -   11. Heat at 99° C. for 5 min and cool down to 4° C. in PCR        machine    -   12. Transfer 50 μl to micro titre plate    -   13. Measure absorbance at 565 nm by plate reader    -   14. Take samples from reaction plate after 24 and 48 hrs and        repeat steps 6-13    -   15. Calculate % of Avicel converted at time 24 and 48 hrs using        formula:

$\begin{matrix}{Y = \frac{\left( {{{OD}\left( {T = {24\mspace{14mu}{or}\mspace{14mu} 48}} \right)} - {{OD}\left( {T = 0} \right)}} \right) \times 100\%}{S \times A}} \\{= \frac{\Delta\;{OD} \times 100}{0.1 \times 10}} \\{= {\Delta\;{OD} \times 100}}\end{matrix}$Y—% of Avicel converted at 24 or 48 hrsS—DNS/glucose calibration slope that is 0.1 for DNS from May 8, 2007 at565 nmA—Avicel concentration at T=0 that is 10 g/L for 1% AvicelReagents:Dinitrosalicylic Acid Reagent Solution (DNS), 1%(Could be stored at 4° C. for several months)

-   -   3,5-dinitrosalicylic acid: 10 g    -   Sodium sulfite: 0.5 g    -   Sodium hydroxide: 10 g    -   Add water to: 1 liter        Calibrate DNS by glucose (use glucose samples with conc.        0,1,2,3,4,5, 6, 7, 8, 9, 10 g/l, calculate the slope [S], for        DNS from May 8, 2007 S=0.1)

24-well PHW Assay

Procedure:

1. Patch all strains to be tested including all controls on selectivemedia plates. Incubate for 2 days

2. Inoculate strains to be tested in 4 ml YPD in 24 well plates(autoclaved) in triplicates. Cover plates with two sticky Rayon Filmsfor Biological Cultures (VWR). Grow with shaking for 2-3 days, 35° C. at225 rpm (attach plates on sticky pads in the fermentation lab shaker)3. Per 24-well plate, prepare substrate mix in a final volume of 100 mL:

Amount to add Substrate/ per 100 mL Concentration Concentration StockSolution Master Mix in Master Mix in PHW assay MS149 Pretreated 8.3 g 4%2% wood (~48% solids) CaCO₃ 0.30 g   3 g/L 1.5 g/L  1M Na citrate(sodium   15 mL 150 mM  75 mM citrate dihydrate) pH 5.4 100XAnti-fungal/    2 mL 2X 1X bacterial mix, Sigma #A5955 Novozyme-188 β-100 ul 0.140 mg/mL 0.070 mg/mL glucosidase (141 mg/mL) dH20 Bring volume— — to 100 mL4. If testing for synergy with other enzymes, aliquot additional enzymesinto appropriate wells (for instance, for synergy with yeast made CBHs,mix purified CBH1 and CBH2 to reach ratio 1:1 and aliquot the mix forthe final concentration 2 mg CBH/g DW PHW). 24 well plates and tips forthis assay don't have to be sterile5. Using 5 mL cut tips, add 2 mL/well of the substrate mix preparedabove to a 24-well assay plate. Use continuous stirring with a magneticstirrer while dispensing the substrate6. Spin cultures to be tested in 24 wp at 3000 rpm for 5 min7. Add 2 mL of supernatants to 24-well assay plate with substrate mixusing multichannel pipette with adjustable spacer for 100-1200 μl(Rainin)8. For negative control, strain M0509 or empty vector strains could beused. For the positive control, dilute Zoomerase to 160 μg/mL (4 mg/g DWPHW) in negative control strain supernatant9. Take T=0 sample by allowing the substrate in the plate to settleeither by gravity or by centrifugation. Then transfer 200 μL ofsupernatant to 96 PCR wp using multichannel pipette with adjustablespacer for 20-300 μl (Rainin). The samples could be frozen at this pointfor future analysis10. Put 24-well assay plate into shaker and incubate at 35° C. at 225rpm (attach plates on sticky pads in the fermentation lab shaker)11. Take subsequent time points, preferably 24 and 48 hours12. For HPLC analysis aliquot 5 μL 10% sulphuric acid into 96 wp withfilters (Millipore, MSGVN2250). Add 100 μl of samples. After filtration(using vacuum in analytical lab), transfer the samples to a totalrecovery HPLC vials for analysis on the H-column. Multichannel pipettewith adjustable spacer for 20-300 μl (Rainin) could be used for transferto make it faster. 96-well collection plate used to collect filteredsamples could be recycled13. Glucose and xylose concentration in the samples also could bemeasured by kits (see separate protocols)

Mini Vials Fermentation Assay

Procedure for Corn Mash:

1) Determine the solids content of the mash by drying it at 105° C. andweighing

2) Weigh liquid corn mash into the 10 mL pressure bottles according tothe desired final % of solids

3) To each bottle add penicillin to final concentration 0.006 mg/mL,urea to final concentration 500 PPM, and water if needed to reach finalweigh 4 g.

4) Add desired enzyme to each bottle.

5) Add yeast cells inoculum to final conc. 0.1 g/L DCW.

6) Cap each bottle and insert the 23 gauge needle into the stopper.

7) Incubate the bottles at desired temperature at 125 rpm.

8) At 72 hours, harvest samples and measure ethanol concentration byHPLC analysis.

Procedure for Corn Flour:

1) Mix corn flour with water according to desired final concentration

2) Add penicillin to final concentration 0.006 mg/mL and urea to finalconcentration 700 PPM

3) Weigh liquid substrate mix into the 10 mL pressure bottles accordingto the desired final % of solids.

4) Add desired enzyme to each bottle.

5) Add yeast cells inoculum to final concentration 0.1 g/L DCW.

6) Cap each bottle and insert the 23 gauge needle into the stopper.

7) Incubate the bottles at desired temperature at 125 rpm.

8) At 72 hours, harvest samples and measure ethanol concentration byHPLC analysis.

Shake Flask Fermentation

Procedure for Corn Mash:

1) Inoculate yeast into 50 mL of YPD and incubate for 15-18 hrs at 35°C. at 200 rpm

2) Spin cell down in 50 mL Falcon tubes, resuspend in 50 mL of water andspin again.

3) Resuspend cells in 10 mL of sterile water and determine dry cellweigh concentration by liquid moister analyzer (Sartorius).

1) Determine the solids content of the mash by drying it at 105° C. andweighing

2) Add mash into shake flasks according to desired final solidsconcentration

3) Add penicillin to final concentration 0.006 mg/mL, urea to finalconc. 500 PPM, and water if needed to reach final weigh 50 g.

4) Add desired enzyme to each flask.

5) Dilute 0.005 g of cells in 1 mL of water and add cells to the flask(0.1 g/L inoculum)

6) Take 1 mL samples at T=24 h, T=48 h and T=72 h. Dilute samples 4× andmeasure ethanol and sugars concentration by HPLC analysis.

Procedure for Corn Flour:

1) Inoculate yeast into 50 mL of YPD and incubate for 15-18 hrs at 35 Cat 200 rpm

2) Spin cell down in 50 mL Falcon tubes, resuspend in 50 mL of water andspin again.

3) Resuspend cells in 10 mL of sterile water and determine dry cellweigh concentration by liquid moister analyzer (Sartorius).

4) Mix corn flour with water according to desired final conc.

5) Add penicillin to final conc. 0.006 mg/mL and urea to final conc. 700PPM

6) Weigh liquid substrate mix into shake flasks according to the desiredfinal % of solids.

7) Add desired enzyme to each flask.

8) Dilute 0.005 g of cells in 1 mL of water and add cells to the flask(0.1 g/L inoculum)

-   -   Take 2 mL samples at T=24 h, T=48 h and T=72 h. Measure ethanol        and sugars concentration by HPLC analysis.

Xylan Assay

1. Prepare a substrate solution: 1,0% Birchwood 4-O-methylglucuronoxylan (Sigma) in 0.05 M Na-citrate buffer, pH 5.0. Homogenize1.0 g in 80 ml buffer at 60° C. and heat to boiling point, on a magneticstirrer. Cool with continued stiffing, cover and stir slowly overnight.Make up to 100 ml with buffer. Store at 4° C. for a maximum of 1 week orfreeze aliquots of e.g. 25 ml at −20° C.2. Aliquot 150 μl of substrate into 96-well PCR plate3. Add 16.7 μl of enzyme containing supernatant4. Incubate at 35° C. for 3 h5. Remove 25 μl of assay sample and mix with 50 μl DNS in a PCR plate6. Boil at 99° C. for 5 min; cool at 4° C.7. Transfer 50 μl to flat bottom corning plate8. Read absorbance at 540 or 565 nm

Xylan Plate Assay

1. Prepare substrate: mix 0.1% Azurine-Crosslinked Xylan (Megazymes)with 1.5% agar in water and autoclave for 20 min

2. Pore substrate on pre-made YPD plates and wait until solid

3. Patch yeast colonies and incubate at 35° C. for 24-48 hrs.

Esterase Assay (for AXE and FAE)

1. Prepare substrate: 1M 4-Nitrophenyl acetate (Sigma N-8130) inmethanol or DMSO

2. Dilute substrate to 1 mM by 50 mM Na-Citrate buffer pH5.4

3. Put 50 μl of enzymes containing yeast supernatants or controls into a96-well analytical plate

4. Add 100 μl 4-Nitrophenyl acetate preheated (35° C.) substrate

5. Read absorbance at 410 nm over a given time course: e.g. 30 min, 1 hrand 2 hours. Incubate sample plate at 35° C. between time points.

6. Reaction can be stopped by adding 100 μl Na₂CO₃ (1 M).

Arabinofuranosidase Assay

1. Prepare substrate: 1M 4-Nitrophenyl α-L-arabinofuranoside (pNPA)(Sigma N-3641) in methanol

2. Dilute substrate to 1 mM by 50 mM Na-Citrate buffer pH5.4

3. Put 20 μl of enzymes containing yeast supernatants or controls into a96-well analytical plate

4. Add 180 μl 4-Nitrophenyl acetate preheated (35° C.) substrate

5. Read absorbance at 405 nm over a given time course: e.g. 30 min, 1 hrand 2 hours Incubate sample plate at 35° C. between time points

6. Reaction can be stopped by adding 100 μl Na₂CO₃ (1 M)

PWC (Pretreated Wet Cake) Assay

1. Prepare substrate mix (70 ml for one 24-well plate): 8 g of 35% PWC(modified distiller's dried grains (MDDG) pretreated at 160 C for 20min), 7 ml 0.5% NaAz, 5.25 ml of 1 M Na Citrate pH5, 0.7 ml of 100×anti-fungal/bacterial mix (Sigma#A5955), and water to final volume 70 ml2. Aliquot purified enzymes into 24-well deep plate in desired amount(under 200 μl)3. Add 2 ml of enzymes containing yeast supernatants or supernatant ofempty strain (no enzymes) as control4. Add 2 ml of substrate mix5. Incubate at 35° C. with shaking for 48 hrs6. Take 200 μl samples at T=0, T=24, T=48 hrs (allow the substrate inthe plate to settle either by gravity or by centrifugation) into 96-wellPCR plate.7. Spin down PCR plate and transfer 100 μL of supernatant to 96-well,0.2 μm filter plate (Fisher: Millipore# MSGVN2250) with 5 μL 10%sulphuric acid added.8. Use filtered sample to measure ethanol and sugars concentration byHPLC.

Xyloglucanase Assay (96-Well Plate)

70 μL of supernatant of 3 day old 2×SC^(−ura) cultures were added to 280μL of 50 mM Na-Acetate buffer (pH 5.0) containing 0.5% AZCL(Azurine-Crosslinked) tamarind xyloglucan (Megazyme catalog # I-AZXYG)in a 96-well deep plate

The plate was incubated in a microtiter plate shaker at 35° C. at 800rpm agitation

Samples of 100 μL were taken at 0, 60 and 180 minutes of incubation into96-well PCR plate spun down at 3000 rpm for 2 min after which 50 μL ofthe supernatant was placed in a fresh 96-well analytical plate and OD at600 nm was measured

Xyloglucanase Plate Assay

Plates containing 1.5% agar+YPD were overlain with 0.1 or 0.5% AZCL(Azurine-Crosslinked) tamarind xyloglucan (Megazyme catalog # I-AZXYG)in 1.5% agar and spotted with 2 μL of overnight yeast culture. Plateswere incubated overnight at 35° C. Blue zone indicated hydrolysis ofsubstrate

Pullulan Assay

1. Add 150 μl of 1% pullulan (in 100 mM NaCitrate buffer pH5.0) to eachwell

2. Mix 16.7 μl of enzyme supernatant

3. Incubate 3 h at 35° C. with shaking (900 rpm)

4. Remove 25 μl of assay sample and mix with 50 μl DNS (the same as instarch assay) in a PCR plate

5. Boil at 99° C. for 5 min; cool at 4° C.

6. Transfer 50 μl to flat bottom corning plate

7. Read absorbance at 565 or 540 nm

Pectin Assay

1. Made 0.1% pectin solution (0.05 g of apple pectin in 50 mL of 100 mMsodium citrate buffer pH 5.0; heat to dissolve)

2. Put 50 μL enzyme containing supernatants into wells of new 96 deepwell plate (5 μL multifect pectinase in M0139 supernatant for total of50 μL)

3. Added 450 μL pectin solution

4. Incubated at 35° C., 900 rpm for 4 hr

5. Aliquot 100 μL DNS (same as in starch assay) into 96-well PCR plate

6. Added 50 μL pectin/supernatants solution to DNS and heated at 99° C.for 5 min followed by cooling down to 4° C.

7. Transferred 50 μL to assay plate (flat-bottomed) and measuredabsorbance at 565 nm or 540 nm

Modified Avicel Assay Protocol:

Procedure:

-   -   Inoculate strains to be tested in 600 ul YPD in deep 96-well        plate. Do 4 repeats for each strain or 4 transformants for each        transformation. Grow with shaking for 3 days at 30° C.    -   1. Spin cells at max speed for 10 min    -   2. Prepare substrate mix:        -   Substrate mix for full 96-well plate, total volume 30 ml:        -   0.6 g Avicel (2%)        -   500 μl 3M Na Ac pH 5.0 (50 mM)        -   1.2 ml 0.5% Na Azide (0.02%)        -   30 μl BGL (Novozyme-188, Sigma)        -   600 μl Zoomerase from 1 mg/ml stock (to get 1 mg/gm of            avicel        -   Add dH2O to 30 ml.    -   3. Add substrate to new deep 96-well plate, 300 μl/well. Shake        between additions, don't let Avicel to settle.    -   4. Add 300 μl of yeast spined supernatant or buffer to the        substrate.    -   5. Take T=0 sample: by multichannel pipette mix the reaction mix        and transfer 100 μl to 96-well PCR plate    -   6. Put deep 96-well reaction plate into shaker at 35° C. and 800        rpm    -   7. Spin 96-well PCR plate with T=0 samples at 2000 rpm for 2 min    -   8. Aliquot 50 μl of DNS into new 96-well PCR plate    -   9. Carefully (without touching pellet) take 25 μl of super from        T=0 spined 96-well PCR plate and mix it into DNS    -   10. Heat at 99° C. for 5 min and cool down to 4° C. in PCR        machine    -   11. Transfer 50 μl to micro titre plate.    -   12. Measure absorbance at 540 nm by plate reader    -   13. Take samples from reaction plate after 2 and 4 hrs and        repeat steps 6-13    -   14. Calculate % of Avicel converted at time 2 and 4 hrs using        formula:

$\begin{matrix}{Y = \frac{\left( {{{OD}\left( {T = {24\mspace{14mu}{or}\mspace{14mu} 48}} \right)} - {{OD}\left( {T = 0} \right)}} \right) \times 100\%}{S \times A}} \\{= \frac{\Delta\;{OD} \times 100}{0.25 \times 10}} \\{= {\Delta\;{OD} \times 40}}\end{matrix}$Y—% of Avicel converted at 24 or 48 hrsS—DNS/glucose calibration slope that is 0.25 for DNS at 540 nmA—Avicel concentration at T=0 that is 10 g/L for 1% Avicel

Reagents:

Dinitrosalicylic Acid Reagent Solution (DNS), 1%

(Could be stored at 4° C. for several months)

-   -   3,5-dinitrosalicylic acid: 10 g    -   Sodium sulfite: 0.5 g    -   Sodium hydroxide: 10 g    -   Add water to: 1 liter        Calibrate DNS by glucose (use glucose samples with conc.        0,1,2,3,4,5, 6, 7, 8, 9, 10 g/l, calculate the slope [S], for        DNS S=0.25)        Concentration Determination of TeCBH1-HgCBM-C and C1CBH2b in        Media by HPLC Analysis.

For determination of the concentration of CBHs produced by strainsexpressing TeCBH1-HgCBM-C(M1111, expressing plasmid pMU1392) and ClCBH2b(M1873), a phenyl reversed phase method was developed on an Agilent 2100HPLC with the MWD detector at 214 and 280 nm. In this method, thepurified CBHs described above were used for generating a standard curvefrom 200—10 μg. The sample was injected onto a phenyl RP column (Tosohphenyl-5PW RP, 4.6 mm×7.5 cm, 10 μm) that was equilibrated at 55° C. in0.1% trifluoracetic acid (TFA) (w/v), 20% acetonitrile. The protein waseluted from the column at 0.75 ml/min using a linear gradient ofacetonitrile with 0.1% TFA (w/v) from 20-60% in 45 minutes. Aftercleaning the column with 95% acetonitrile/TFA, the column wasre-equilibrated. To determine the concentration of TeCBH1-HgCBM-C andC1CBH2b produced in media by various strains, the peak area of thesample was compared to the standard curve generated from the peak areasof the purified CBHs (μg/μL injected).

Purification of TeCBH1-HgCBM-C and C1CBH2b for Protein Standards in theHPLC Assay.

1 or 1.5 liter of YPD medium was inoculated with a 10% volume of anovernight pre-culture of the strain producing CBH1 or CBH2 (M1111,expressing plasmid pMU1392 and M1873, respectively). The cultures weregrown with shaking (210 rpm) at 30° C. After 3 days of cultivation thesupernatants were harvested by removing the cells by centrifugation. Thesupernatants were concentrated and changed into 50 mM sodium acetate (pH5) with a 10 kDa cut-off Pellicon PTGC membrane (Millipore). The CBH1sample was loaded into DEAE Sepharose FF column equilibrated with 50 mMsodium acetate, pH 5.0. The bound CBH1 was eluted with linear saltgradient of from 0 to 0.35 M NaCl. The elution volumes were 15 and 20column volumes. The fractions were tested for CBH1 activity with MULacby incubating 10 μl sample with 90 μl 2 mM MULac in 50 mM NaAc (pH 5.0),in ambient temperature for 20 minutes and stopping the reaction with 0.5M Na₂CO₃. The fluorescence was measured with a Varioscan (ThermoLabsystems) microtiter plate reader (ex. 355 nm and em. 460 nm). TheCBH1 proteins were visualized on SDS-PAGE and the fractions containing asingle band were pooled and changed into 50 mM sodium acetate (pH 5)using 20 ml spin concentrators, 10 kDa MWCO (Vivaspin, VivascienceGmbH). A second step was then carried out in the purification where a 5ml GE phenyl HR column was utilized to further remove media components.In this procedure, the column was equilibrated with 25 mM sodiumacetate, 1.2 M ammonium sulfate, pH 5 Ammonium sulfate was added to thesample to bring the concentration in the buffer to 1.2 M and thismaterial was injected onto the column. The protein was eluted with alinear gradient of 25 mM sodium acetate, pH 5 and fractions that wereactive on MULac were pooled. Purity was assessed by SDS-PAGE andconcentration was determined by absorbance at 280 nm using thetheoretical absorptivity value. C1CBH2b was purified using the samechromatography steps, DEAE anion exchange followed by phenyl HIC. Inthis purification, C1CBH2b is found in the flow through of the DEAE stepand was eluted from the phenyl HIC column within the decreasing ammoniumsulfate gradient. Active fractions were determined using a 1% Avicelhydrolysis assay at pH 5.0 as described above. Purity and concentrationdetermination were determined as described above.

PHW Assay

1. Prepare substrate mix (100 mL per one 24-well plate): 8.3 g ofpretreated wood (48% of solids), 20 ml of 1M Na Citrate pH4.8, 2 ml of100× anti-fungal/bacterial mix (Sigma#A5955), and water to final volume100 ml. In some assay 0.222 ml of commercial glucoamylase (ABEnzymes#EL2008044L 63 ml/ml) is added (heat treated to remove sideactivities)2. Add purified enzymes into wells of 24-well deep plate (under 200 μl)3. Add 2 mL of enzymes containing yeast supernatants and empty strainsupernatant as control4. Using cut 5 ml tips, add 2 ml/well of the substrate mix to enzymes.Use continuous stirring with a magnetic stirrer while dispensing thesubstrate5. Incubate 24-well reaction plate at 38° C. and 250 rpm6. Take 200 μl samples at T=0, T=24, T=48 hrs (allow the substrate inthe plate to settle either by gravity or by centrifugation) into 96-wellPCR plate7. Spin down PCR plate and transfer 100 μL of supernatant to 96-well,0.2 μm filter plate (Fisher: Millipore# MSGVN2250) with 5 μL 10%sulphuric acid added8. Use filtered sample to measure ethanol and sugars concentration byHPLC

Paper Sludge Assay

1. Prepare substrate mix (100 mL per one 24-well plate): 10.5 g of papersludge (38% of solids), 40 ml of 1M Na Citrate pH5.2, 2 ml of 100×anti-fungal/bacterial mix (Sigma#A5955), and water to final volume 100ml. In some assays 0.222 ml of commercial thermostable β-glucosidase (ABEnzymes 63 ml/ml) is added (heat treated to remove side activities)2. Add purified enzymes into wells of 24-well deep plate (under 200 μl)3. Add 2 mL of enzymes containing yeast supernatants and empty strainsupernatant as control4. Using cut 5 ml tips, add 2 ml/well of the substrate mix to enzymes.Use continuous stirring with a magnetic stirrer while dispensing thesubstrate5. Incubate 24-well reaction plate at 35° C. and 250 rpm6. Take 200 μl samples at T=0, T=24, T=48 hrs (allow the substrate inthe plate to settle either by gravity or by centrifugation) into 96-wellPCR plate7. Spin down PCR plate and transfer 100 μL of supernatant to 96-well,0.2 μm filter plate (Fisher: Millipore# MSGVN2250) with 5 μL 10%sulphuric acid added8. Use filtered sample to measure ethanol and sugars concentration byHPLC.1—Napthyl-Acetate Esterase Assay1. Inoculate SC or YPD medium with the stain to be tested and incubateon a rotary shaker.2. Remove the cells by centrifugation.3. Set up the reaction as follows in a 96 well plate:

-   -   88 μL Citrate buffer (50 mM, pH 5.0)*    -   10 μL Supernatant    -   2 μL 1-naphtyl-acetate in ethanol (500 mM)**    -   100 μL Total        * (Phosphate buffer can also be used but Acetate buffers cause a        precipitate)        **(Sigma 46010)        4. Incubate for 5-30 min at 35° C. The incubation time depend on        the level of activity.        5. Stop the reaction by adding 100 μl 0.01% Fast Corrinth V salt        solution.        6. Read 100 μL at 535 nm

50 mM Citrate Buffer pH 5.0

1M Citric acid 20.5 mL 1M Na-citrate 29.5 mL This is 50 mL 1M CitratePhosphate buffer (pH 5.0). Dilute to appropriate concentration withwater.

500 mM 1-Naphtyl-Acetate (Mr 186 g/mol)

1-naphtyl-acetate 0.0931 g Ethanol (100%) 1000 μl (make fresh batch eachday)

Fast Corrinth V Salt Solution (Sigma 227366)

Fast Corrinth V salt (0.01%) 0.001 g Tween 20 (10%) 1 mL 1M Na-Acetatebuffer pH 4.4 9 mL 10 mLNB: Make this Solution Fresh Each Day and Keep in a Dark Bottle—Use SameDay, Very Light Sensitive.

1-Naphtol (for Standard Curve) (Sigma 31097)

-   -   Prepare a 1 g/L 1-naphtol solution in the buffer used for the        assay to set the standard curve.    -   Set the standard cure between 0.025 g/L and 0.4 g/L    -   Alpha-galactosidase Activity Assay using NpGal

-   Reference: Margolles-Clark et al. 1996. Eur J Biochem. 240: 104-111.

-   1. Prepare solutions as indicated below

-   2. Patch colonies to be screened on selection plates and incubate at    30-35° C. for 48 h

-   3. Inoculate 600 μl YPD in 96 well plate and incubate at 35° C. with    800 rpm shaking for 48-72 h

-   4. Spin cells for 2 min at 2500 rpm

-   5. Place 20 μl supernatant into a 96 well plate

-   6. Add 180 μl NpGal preheated (35° C.) substrate

-   7. Incubate for given time course at 35° C.: e.g. 30 min, 1 hr and 2    hours (may have to go overnight according to some enzymes in    literature)

-   8. Read absorbance at 405 nm over a given time course. Incubate    sample plate at 35° C. between time points

-   9. Stop reaction by adding 100 μl Na₂CO₃ (1 M)    1 mM p-nitrophenyl-α-D-galactopyranoside (NpGal) (Sigma N0877) 301.3    g/mol)    -   Make a 1M Stock=0.151 g in 500 μl methanol or DMSO    -   1 mM Stock=10 μl of 1M stock in 9.99 ml citrate buffer        Citrate Buffer (0.05 M pH 5.4) 1L    -   0.1 M Citric acid: 21.01 g citric acid in 1000 ml H₂O    -   0.1 M Sodium citrate: 29.41 g of C₆H₅O₇Na₃.2H₂O in 1000 ml H₂O    -   20.5 ml of citric acid+29.5 ml of sodium citrate, add dH₂O to a        total of 100 ml

INCORPORATION BY REFERENCE

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A recombinant yeast host cell comprising a heterologouspolynucleotide encoding a polypeptide comprising an amino acid sequenceat least 90% identical to the amino acid sequence of SEQ ID NO: 445; andat least one heterologous polynucleotide encoding a polypeptidecomprising an amino acid sequence at least 90% identical to the aminoacid sequence selected from the group consisting of: SEQ ID NO: 443, SEQID NO: 496, SEQ ID NO: 498 and SEQ ID NO: 504 wherein the recombinantyeast host cell expresses the polypeptides.
 2. The recombinant yeasthost cell of claim 1, wherein the heterologous polynucleotide encodes apolypeptide comprising an amino acid sequence at least 95% identical tothe amino acid sequence of SEQ ID NO: 445 and at least one heterologouspolynucleotide encoding a polypeptide comprising an amino acid sequenceat least 95% identical to the amino acid sequence selected from thegroup consisting of: SEQ ID NO: 443, SEQ ID NO: 496, SEQ ID NO: 498 andSEQ ID NO:
 504. 3. The recombinant yeast host cell of claim 2, whereinthe heterologous polynucleotide encodes a polypeptide comprising theamino acid sequence of SEQ ID NO: 445 and at least one heterologouspolynucleotide encoding a polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 443, SEQ ID NO: 496,SEQ ID NO: 498 and SEQ ID NO:
 504. 4. The recombinant yeast host cell ofclaim 1, wherein the yeast strain is a Saccharomyces cerevisiae strain.5. The recombinant yeast host cell of claim 1, further comprising one ormore additional heterologous polynucleotides encoding one or moreadditional polypeptides comprising an amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof: SEQ ID NO: 287, SEQ ID NO: 450, SEQ ID NO: 451, and SEQ ID NO: 452.6. The recombinant yeast host cell of claim 5, wherein the one or moreadditional heterologous polynucleotides encoding one or more additionalpolypeptides comprises an amino acid sequence at least 95% identical toan amino acid sequence selected from the group consisting of: SEQ ID NO:287, SEQ ID NO: 450, SEQ ID NO: 451, and SEQ ID NO:
 452. 7. Therecombinant yeast host cell of claim 5, wherein the one or moreadditional heterologous polynucleotides encoding one or more additionalpolypeptides comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 287, SEQ ID NO: 450, SEQ ID NO: 451, and SEQID NO:
 452. 8. The recombinant yeast host cell of claim 5, wherein theyeast strain is a Saccharomyces cerevisiae strain.
 9. The recombinantyeast host cell of claim 1, wherein the host cell further expresses axylose isomerase.
 10. The recombinant yeast host cell of claim 9,wherein the host cell is capable of fermenting a pentose sugar.
 11. Therecombinant yeast host cell of claim 10, wherein the pentose sugar isxylose or arabinose.
 12. The recombinant yeast host cell of claim 9,wherein the xylose isomerase is isolated from an organism selected fromthe group consisting of: Piromyces, Bacterioides thetaiotaomicron,Chitinophaga pinensis, Parabacteroides distasonis, and combinationsthereof.
 13. The recombinant yeast host cell of claim 1, furthercomprising one or more additional heterologous polynucleotides encodingone or more additional polypeptides comprising an amino acid sequence atleast 90% identical to an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ IDNO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460,SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, and SEQID NO:
 465. 14. The recombinant yeast host cell of claim 13, furthercomprising one or more additional heterologous polynucleotides encodingone or more additional polypeptides comprising an amino acid sequence atleast 95% identical to an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ IDNO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460,SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, and SEQID NO:
 465. 15. The recombinant yeast host cell of claim 14, furthercomprising one or more additional heterologous polynucleotides encodingone or more additional polypeptides comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 453, SEQ ID NO: 454,SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ IDNO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463,SEQ ID NO: 464, and SEQ ID NO:
 465. 16. The recombinant yeast host cellof claim 13, wherein the yeast strain is a Saccharomyces cerevisiaestrain.
 17. The recombinant yeast host cell of claim 1, furthercomprising one or more additional heterologous polynucleotides encodingone or more additional polypeptides comprising an amino acid sequence atleast 90% identical to the amino acid sequence of a polypeptide selectedfrom the group consisting of: SEQ ID NO: 447, SEQ ID NO: 448, and SEQ IDNO:
 449. 18. The recombinant yeast host cell of claim 1, wherein thequantity of ethanol that is produced at 35.degree. C. on a substrate ofcorn mash, corn flour or combinations thereof is at least about 125grams per liter in 72 hours.
 19. The recombinant yeast host cell ofclaim 1, wherein the quantity of ethanol that is produced at 35.degree.C. on a substrate of corn flour is at least about 1.9 grams per literper hour.
 20. The recombinant yeast host cell of claim 1, wherein thequantity of ethanol that is produced at 35.degree. C. on a substrate ofcorn mash is at least about 2.2 grams per liter per hour.
 21. Therecombinant yeast host cell of claim 1, where the ethanol is produced ata temperature of at least about 35.degree. C.
 22. The recombinant yeasthost cell of claim 17, comprising one or more copies of each of theheterologous polynucleotides encoding the polypeptides comprising aminoacid sequences at least 90% identical to the amino acid sequences of thepolypeptides encoded by: SEQ ID NO: 447, SEQ ID NO: 448, and SEQ ID NO:449.
 23. The recombinant host cell of claim 17, comprising four or morecopies of the heterologous polynucleotide encoding a polypeptidecomprising an amino acid sequence at least 90% identical to the aminoacid sequence of SEQ ID NO: 445 and one or more copies of each of theheterologous polynucleotides encoding the polypeptides comprising aminoacid sequences at least 90% identical to the amino acid sequences of thepolypeptides encoded by: SEQ ID NO: 447, SEQ ID NO: 448, and SEQ ID NO:449.
 24. The recombinant yeast host cell of claim 17, further comprisinga down-regulation of at least one gene encoding an endogenousglycerol-producing enzyme selected from the group consisting of:glycerol 3-phosphate dehydrogenase 1 and glycerol-3 phosphatedehydrogenase
 2. 25. The recombinant host cell of claim 1, comprisingtwo or more copies of the heterologous polynucleotide encoding apolypeptide comprising an amino acid sequence at least 90% identical tothe amino acid sequence of SEQ ID NO:
 445. 26. The recombinant host cellof claim 1, comprising four or more copies of the heterologouspolynucleotide encoding a polypeptide comprising an amino acid sequenceat least 90% identical to the amino acid sequence of SEQ ID NO: 445.